Biopharmaceutical compositions

ABSTRACT

The present disclosure relates to compositions, for treating interleukin 5 (IL-5) mediated diseases, and related methods.

FIELD OF THE DISCLOSURE

The present disclosure relates to compositions, for treating interleukin5 (IL-5) mediated diseases, and related methods.

BACKGROUND OF THE DISCLOSURE

IL-5 plays a role in a number of different diseases such as asthma,severe eosinophilic asthma, severe asthma, uncontrolled eosinophilicasthma, eosinophilic asthma, sub-eosinophilic asthma, chronicobstructive pulmonary disease, eosinophilic granulomatosis withpolyangiitis, hypereosinophilic syndrome, nasal polyposis, bullouspemphigoid and eosinophilic esophagitis. These serious diseases affecthundreds of millions of people world wide.

Mepolizumab is a monoclonal antibody that binds to soluble IL-5 andblocks the soluble IL-5 from binding to its receptor. The structure ofIL-5 is indicative of a secreted protein and there is no evidence of anymembrane-bound forms of IL-5 on any cell types. Thus, Fc effectorfunctions are not part of the mepolizumab mechanism of action. Based onthe mechanism of action and pharmacokinetic properties of mepolizumab,there are two functional domains involved in the biological activity ofthis monoclonal antibody. These are a) binding to IL-5 in complementarydetermining region (CDR) which provides the mechanism of action; and b)binding to neonatal Fc receptor (FcRn) receptor in Fc region, whichdetermines the half-life. Through extensive characterization studiesperformed throughout the development of the product, it has beendetermined that deamidation, oxidation, and aggregation are criticalquality attributes of mepolizumab. Importantly, it has been found thatspecific levels of these variants must be maintained to ensureappropriate biological function.

Thus, a need exists for compositions suitable for maintaining thebiological function of mepolizumab and for treating IL-5 mediateddisease. Such compositions and related methods are provided by thepresent disclosure.

SUMMARY OF THE DISCLOSURE

One aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤80% acidic antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤80% acidic antibody variants and≤20% aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variant atN31 of the light chain amino acid sequence; and ≤20% aggregated antibodyvariants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤55% oxidised antibodyvariants at M64 of the heavy chain amino acid sequence; ≤3% oxidisedvariant at W52 of the heavy chain amino acid sequence; and ≤20%aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤35% deamidated antibodyvariants at N386 of the heavy chain amino acid sequence; and ≤20%aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤35% deamidated antibodyvariants at N386 of the heavy chain amino acid sequence; ≤55% oxidisedantibody variants at M64 of the heavy chain amino acid sequence, M254 ofthe heavy chain amino acid sequence, M430 of the heavy chain amino acidsequence; ≤3% oxidised antibody variants at W52 of the heavy chain aminoacid sequence; and ≤20% aggregated antibody variants.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.8 to 7.2, wherein the buffering agent ishistidine, phosphate, citric acid, citrate or a salt thereof, whereinthe purified preparation comprises the isoforms represented by peak 65,peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1,wherein the antibody comprises a heavy chain amino acid sequence havingat least 90% identity to the amino acid sequence of SEQ ID NO: 1 and alight chain amino acid sequence having at least 90% identity to theamino acid sequence of SEQ ID NO: 2, and wherein the antibody isproduced by a Chinese Hamster Ovary cell.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.8 to 7.2, wherein the buffering agent isphosphate or a salt thereof, wherein the purified preparation comprisesthe isoforms represented by peak 65, peak 78, peak 88, peak 92, the mainpeak and peak 112 shown in FIG. 1, wherein the antibody comprises aheavy chain amino acid sequence having at least 90% identity to theamino acid sequence of SEQ ID NO: 1 and a light chain amino acidsequence having at least 90% identity to the amino acid sequence of SEQID NO: 2, and wherein the antibody is produced by a Chinese HamsterOvary cell.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) a main form of the antibody comprisinggreater than, or equal to, 50% of the protein in the composition asmeasured using capillary isoelectric focusing of the composition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) acidicforms of the antibody comprising about 20% to about 45% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) a basicform of the antibody comprising about 1% to about 15% of the protein inthe composition as measured using capillary isoelectric focusing of thecomposition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; c) acidic formsof the antibody comprising about 20% to about 45% of the protein in thecomposition as measured using capillary isoelectric focusing of thecomposition; and d) a basic form of the antibody comprising about 1% toabout 15% of the protein in the composition as measured using capillaryisoelectric focusing of the composition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) deamidated forms of the antibodycomprising at least one selected from the group consisting of a heavychain amino acid residue deamidated at asparagine 299, a heavy chainamino acid residue deamidated at asparagine 317, a heavy chain aminoacid residue deamidated at asparagine 386 and a light chain amino acidresidue deamidated at asparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) oxidized forms of the antibody comprisingat least one selected from the group consisting of a heavy chain aminoacid residue oxidized at tryptophan 52, a heavy chain amino acid residueoxidized at methionine 64, a heavy chain amino acid residue oxidized atmethionine 82, a heavy chain amino acid residue oxidized at methionine85, a heavy chain amino acid residue oxidized at cysteine 222, a heavychain amino acid residue oxidized at methionine 254, a heavy chain aminoacid residue oxidized at methionine 360, a heavy chain amino acidresidue oxidized at methionine 430, a light chain amino acid residueoxidized at methionine 4 and light chain amino acid residue oxidized atcysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) deamidated forms of the antibody comprising atleast one selected from the group consisting of a heavy chain amino acidresidue deamidated at asparagine 299, a heavy chain amino acid residuedeamidated at asparagine 317, a heavy chain amino acid residuedeamidated at asparagine 386 and a light chain amino acid residuedeamidated at asparagine 31; and c) oxidized forms of the antibodycomprising at least one selected from the group consisting of a heavychain amino acid residue oxidized at tryptophan 52, a heavy chain aminoacid residue oxidized at methionine 64, a heavy chain amino acid residueoxidized at methionine 82, a heavy chain amino acid residue oxidized atmethionine 85, a heavy chain amino acid residue oxidized at cysteine222, a heavy chain amino acid residue oxidized at methionine 254, aheavy chain amino acid residue oxidized at methionine 360, a heavy chainamino acid residue oxidized at methionine 430, a light chain amino acidresidue oxidized at methionine 4 and light chain amino acid residueoxidized at cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) oxidized forms of the antibody comprising a heavy chainamino acid residue oxidized at methionine 64.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) oxidized forms of the antibody comprising a heavy chainamino acid residue oxidized at methionine 64; and c) deamidated forms ofthe antibody comprising a light chain amino acid residue deamidated atasparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; and b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; and b) oxidized forms of the antibody comprising at least oneselected from the group consisting of a heavy chain amino acid residueoxidized at tryptophan 52, a heavy chain amino acid residue oxidized atmethionine 64, a heavy chain amino acid residue oxidized at methionine82, a heavy chain amino acid residue oxidized at methionine 85 and alight chain amino acid residue oxidized at methionine 4.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31; and c) oxidized forms ofthe antibody comprising at least one selected from the group consistingof a heavy chain amino acid residue oxidized at tryptophan 52, a heavychain amino acid residue oxidized at methionine 64, a heavy chain aminoacid residue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85 and a light chain amino acid residue oxidizedat methionine 4.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of an amino terminal pyroglutamate residue at aminoacid residue 1, a carboxy terminal glycine amino acid residue at aminoacid residue 448, a deamidated asparagine residue at position 299, adeamidated asparagine residue at position 317, a deamidated asparagineresidue at position 386, a oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine at position 222, an oxidized methionine at position254, an oxidized methionine at position 360 and an oxidized methionineresidue at position 430; and b) a modified form of the antibody lightchain amino acid sequence shown in SEQ ID NO: 2 comprising at least oneamino acid residue modification selected from the group consisting of adeamidated asparagine residue at amino acid residue 31, an oxidizedmethionine residue at position 4 and an oxidized cysteine at position220.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated asparagine residue at position 299,a deamidated asparagine residue at position 317, a deamidated asparagineresidue at position 386, an oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine at position 222, an oxidized methionine at position254, an oxidized methionine at position 360, and an oxidized methionineresidue at position 430; and b) a modified form of the antibody lightchain amino acid sequence shown in SEQ ID NO: 2 comprising at least oneamino acid residue modification selected from the group consisting of adeamidated asparagine residue at amino acid residue 31, an oxidizedmethionine residue at position 4 and an oxidized cysteine at position220.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated asparagine residue at position 299,a deamidated asparagine residue at position 317 and a deamidatedasparagine residue at position 386; and b) a modified form of theantibody light chain amino acid sequence shown in SEQ ID NO: 2comprising a deamidated asparagine residue at amino acid residue 31.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of an oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine at position 222, an oxidized methionine at position254, an oxidized methionine at position 360, and an oxidized methionineresidue at position 430; and b) a modified form of the antibody lightchain amino acid sequence shown in SEQ ID NO: 2 comprising at least oneselected from the group consisting of an oxidized methionine residue atposition 4 and an oxidized cysteine at position 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) a main form of the antibody comprisinggreater than, or equal to, 20% of the protein in the composition asmeasured using capillary isoelectric focusing of the composition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 20% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) acidicforms of the antibody comprising up to about 80% of the protein in thecomposition as measured using capillary isoelectric focusing of thecomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Representative capillary isoelectric focusing (cIEF)electropherogram of a reference standard (RS) composition comprisingmepolizumab.

FIG. 2. Representative cIEF electropherograms of a reference standardcomposition comprising mepolizumab (control) and different batches ofthe compositions comprising mepolizumab subjected to three days of pH9.0 forced degradation.

FIG. 3. Representative full view size exclusion chromatography (SEC)chromatogram of a RS composition comprising mepolizumab.

FIG. 4. Representative expanded view SEC chromatogram of a RScomposition comprising mepolizumab.

FIG. 5. Representative SEC-multi-angle light scattering (MALS)chromatogram of a RS composition comprising mepolizumab.

FIG. 6. Representative SEC-MALS chromatogram of a batch of thecomposition comprising mepolizumab.

FIG. 7. Representative SEC Chromatograms of a RS composition comprisingmepolizumab and for different batches of pH 3.5 stressed compositioncomprising mepolizumab at Day 7.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides compositions, for treating interleukin 5(IL-5) mediated diseases, and related subject matter.

The term “asthma” as used herein means an inflammatory disease of theairways characterized by reversible airflow obstruction andbronchospasm. Common symptoms include wheezing, coughing, chesttightness, and shortness of breath.

In the methods of the disclosure “asthma” may be “severe eosinophilicasthma.” Subjects with severe eosinophilic asthma may have asthma andblood eosinophils greater than or equal to 300 eosinophils per μL ofblood in the past 12 months. Subjects with severe eosinophilic asthmamay meet, one or more of, the critera described in Table 1.

TABLE 1 A subject has severe eosinophilic asthma if they meet thefollowing criteria: 1) The subject has clinical features of severerefractory asthma similar to those indicated in the American ThoracicSociety Workshop on Refractory Asthma (162 Am. J. Respir. Crit. CareMed. 2341 (2000) for ≥12 months. 2) The subject has a well-documentedrequirement for regular treatment with high dose ICS (inhaledcorticosteroids) (i.e., ≥880 μg/day fluticasone propionate or equivalentdaily), with or without maintenance OCS (oral corticosteroids), in thepast 12 months. 3) The subject has a well-documented requirement forcontroller medication, e.g., long- acting beta-2-agonist, leukotrienereceptor antagonist or theophylline in the past 12 months. 4) Thesubject has persistent airflow obstruction as indicated by apre-bronchodilator FEV₁ <80% predicted recorded or peak flow diurnalvariability of >20% on 3 or more days. 5) The subject has airwayinflammation which is likely to be eosinophilic in nature as indicatedby one of the following characteristics at present or documented in theprevious 12 months: An elevated peripheral blood eosinophil level of≥300/μL that is related to asthma or Sputum eosinophils ≥3% or Exhalednitric oxide ≥50 ppb or Prompt deterioration of asthma control (based ondocumented clinical history or objective measures) following a ≤25%reduction in regular maintenance dose of inhaled or oral corticosteroiddose in the previous 12 months 8) The subject has a previously confirmedhistory of two or more asthma exacerbations requiring treatment withoral or systemic corticosteroids in the prior 12 months prior, despitethe use of high-dose ICS and additional controller medication. Forsubjects receiving maintenance OCS with high-dose ICS plus controller,the OCS treatment for exacerbations had to be a two-fold or greaterincrease in the dose of OCS. 9) The subject has asthma as documented byeither: Airway reversibility (FEV₁ ≥12% and 200 mL) at present ordocumented in the previous 12 months or Airway hyper-responsiveness(provocative concentration causing a 20% fall in FEV₁ of methacholine <8mg/mL or provocative dose causing a 20% fall in FEV₁ of histamine <7.8μmol) documented in the prior 12 months or Airflow variability in clinicFEV₁ ≥20% between two examinations documented in the prior 12 months(FEV₁ recorded during an exacerbation is not valid) or Airflowvariability as indicated by >20% diurnal variability in peak flowobserved on 3 or more days.Importantly, subjects with severe eosinophilic asthma according to thesecriteria may have less than 150 eosinophils per μL of blood at theinitiation of treatment. Mepolizumab is a monoclonal antibody comprisingthe heavy chain amino acid sequence shown in SEQ ID NO: 1 and the lightchain amino acid sequence shown in SEQ ID NO: 2. Mepolizumab, andantigen binding proteins, in particular antibody molecules, comprisingthe heavy chain CDRs and light chain CDRs of mepolizumab, may be used totreat severe eosinophilic asthma according to the methods of thedisclosure. For example, mepolizumab, or related antigen bindingproteins, may be indicated for add-on maintenance treatment of severeeosinophilic asthma, as identified by blood eosinophils greater than orequal to 300 cells/μL in the past 12 months and/or blood eosinophilsgreater than or equal to 150 cells/μL at initiation of treatment and/orblood eosinophils less than 150 cells/μL at initiation of treatment, inpatients. Alternatively, mepolizumab, or related antigen bindingproteins, may be indicated for add-on maintenance treatment of severeeosinophilic asthma, as identified by blood eosinophils greater than orequal to 300 cells/μL in the past 12 months and/or blood eosinophilsgreater than or equal to 150 cells/μL at initiation of treatment, inpatients. Mepolizumab, or related antigen binding proteins, may beindicated for add-on maintenance treatment of severe eosinophilicasthma, as identified by blood eosinophils greater than or equal to 300cells/μL in the past 12 months and/or blood eosinophils less than 150cells/μL at initiation of treatment, in patients. Such patients may beaged 12 years and older. Mepolizumab treatment may reduce exacerbationsof asthma in patients (e.g., patients with an exacerbation history). Themethods of the disclosure may be used when mepolizumab treatment isindicated (i.e., such treatment with mepolizumab may be combined withthe methods of the disclosure). Treatment with mepolizumab can:a) Produce a reduction in exacerbation frequency. Compared with placebo,treatment with mepolizumab, such as 100 mg per subject administeredsubcutaneously or 75 mg per subject administered intravenously, canreduce the rate of 1) clinically significant exacerbations, 2)exacerbations requiring hospitalization or ED visits, and 3)exacerbations requiring hospitalization. This benefit may potentiallylead to reductions in morbidity and fatal events due to asthma.b) Produce a reduction in daily OCS dose: Treatment with mepolizumab,such as 100 mg per subject administered subcutaneously or 75 mg persubject administered intravenously, may allow subjects to reduce theirdaily dose of concomitant corticosteroid without experiencing loss ofasthma control. Subjects treated with mepolizumab may achieve a medianpercentage reduction of 50% from baseline in daily oral corticosteroid(OCS) dose versus 0% for those treated with placebo. In addition, 54% ofsubjects treated with mepolizumab may achieve a reduction of OCS dose to5.0 mg compared with 32% of subjects treated with placebo (p=0.025).c) Produce an improvement in lung function: Clinically relevant changesin pre- and post-bronchodilator FEV₁ may be demonstrated withmepolizumab treatment, such as 100 mg per subject administeredsubcutaneously or 75 mg per subject administered intravenously, comparedwith placebo. Any improvements in lung function are of particularclinical importance in this population of subjects as most are onmaximal asthma therapy including high dose ICS (inhaled corticosteroids)and/or OCS plus a controller medication.d) Produce an improvement in asthma control: Statistically significantand clinically relevant improvements may be observed in ACQ-5 withmepolizumab, such as 100 mg per subject administered subcutaneously or75 mg per subject administered intravenously, compared with placebo,indicating subjects may achieve asthma control with the addition ofmepolizumab to their existing asthma treatment.e) Produce an improvement in quality of life: Statistically significantand clinically relevant changes in SGRQ scores may be demonstrated withmepolizumab, such as 100 mg per subject administered subcutaneously or75 mg per subject administered intravenously, compared with placebo.Subjects may experience marked improvement in asthma symptoms andability of perform daily activities.f) Produce a persistence of efficacy and pharmacodynamic effect: Over aperiod of 32- and/or 52-week treatment durations, a sustained reductionin asthma exacerbations and blood eosinophils, and improvements in lungfunction, asthma control, and quality of life with no development oftolerance may be observed.andg) Produce a reduction in blood eosinophils. Treatment with compositionscomprising mepolizumab, such as 100 mg of mepolizumab per subjectadministered subcutaneously or 75 mg per subject administeredintravenously, may result in rapid reduction of blood eosinophils(approximately 80% by the first assessment at Week 4 after initialtreatment; e.g., from 250-290 cells/μL to 40-60 cells/μL etc.).

In the methods of the disclosure “asthma” may be “severe asthma.”Subjects with severe asthma meet the definition of severe asthmadescribed in the European Respiratory Society/American Thoracic Society(ERS/ATS) Guidelines for severe asthma. Thus, severe asthma is asthmawhich requires treatment with guideline suggested medications for GlobalInitiative for Asthma (GINA) steps 4-5 asthma (high dose inhaledcorticosteroids [ICS] plus long acting beta2-agonist [LABA] orleukotriene modifier/theophylline) for the previous year, or systemiccorticosteroids (CS) for >=50% of the previous year to maintain controlof the subject's asthma. Treatment with compositions comprisingmepolizumab may be used to treat severe asthma according to the methodsof the disclosure.

In the methods of the disclosure “asthma” may be “uncontrolledeosinophilic asthma.” Subjects with uncontrolled eosinophilic asthmameet the criteria described in Table 2.

TABLE 2 A subject has uncontrolled eosinophilic asthma if they meet thefollowing criteria: 1) The subject has a history of diagnosed asthma forat least the prior 12 months. 2) The subject has been prescribed dailyuse of medium-dose or high- dose ICS (inhaled corticosteroid) plus LABA(long-acting beta agonists) for at least the prior 12 months. 3) Thesubject's dose of other asthma controller medications must be stable forat least the prior 30 days. 4) The subject has at least 2 documentedasthma exacerbations in the prior 12 months that required use of asystemic corticosteroid burst.Treatment with compositions comprising mepolizumab may be used to treatuncontrolled eosinophilic asthma according to the methods of thedisclosure.

In the methods of the disclosure “asthma” may be “eosinophilic asthma.”Subjects with uncontrolled eosinophilic asthma meet the criteriadescribed in Table 3.

TABLE 3 A subject has eosinophilic asthma if they meet the followingcriteria: 1) The patient has a previous diagnosis of asthma. 2) Thepatient has had at least 1 asthma exacerbation requiring oral,intramuscular (im), or intravenous (iv) corticosteroid use for at least3 days in the prior 12 months. 3) The patient has a current bloodeosinophil level of at least 400/μl. 4) The patient has airwayreversibility of at least 12% to beta-agonist administration. 5) Thepatient has an ACQ score of at least 1.5. 6) The patient is takinginhaled fluticasone at a dosage of at least 440 μg, or equivalent,daily. Chronic oral corticosteroid use (no more than 10 mg/dayprednisone or equivalent) is allowed. The patient's baseline asthmatherapy regimen (including, but not limited to, inhaled corticosteroids,oral corticosteroids up to a maximum dose of 10 mg prednisone daily orequivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, orcromolyn) must be stable for the prior 30 days.

In the methods of the disclosure “asthma” may be “sub-eosinophilicasthma.” Subjects with uncontrolled eosinophilic asthma meet thecriteria described in Table 4.

TABLE 4 A subject has sub-eosinophilic asthma if they meet the followingcriteria: 1) The patient has a previous diagnosis of asthma. 2) Thepatient has had at least 1 asthma exacerbation requiring oral,intramuscular (im), or intravenous (iv) corticosteroid use for at least3 days in the prior 12 months. 3) The patient has a current bloodeosinophil level of less than 400/μl. 4) The patient has airwayreversibility of at least 12% to beta-agonist administration. 5) Thepatient has an ACQ score of at least 1.5. 6) The patient is takinginhaled fluticasone at a dosage of at least 440 μg, or equivalent,daily. Chronic oral corticosteroid use (no more than 10 mg/dayprednisone or equivalent) is allowed. The patient's baseline asthmatherapy regimen (including, but not limited to, inhaled corticosteroids,oral corticosteroids up to a maximum dose of 10 mg prednisone daily orequivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, orcromolyn) must be stable for the prior 30 days.Treatment with compositions comprising mepolizumab may be used to treatsub-eosinophilic asthma and may also be used to treat sub-eosinophilicasthma according to the methods of the disclosure.

The term “bullous pemphigoid” (BP) as used herein means an acute orchronic autoimmune skin disease, involving the formation of blisters,more appropriately known as bullae, at the space between the skin layersepidermis and dermis. BP is the most common autoimmune blistering skindisease. It characteristically affects the elderly (>70 years) with anannual incidence of 5 to 35 per million The incidence of BP isdramatically increasing with an average of 17% per year. BP often startswith extremely pruritic skin lesions resembling eczema or urticariabefore vesicles and blisters arise. In 10-30% of patients, BP alsoinvolves the oral mucosa. Disease severity can be determined by means ofthe autoimmune bullous skin disorder intensity score (ABSIS) thatevaluates the involved area as well as the disease activity. The diseaseis due to an autoimmune response to structural components of junctionaladhesion complexes leading to the damage of the dermal-epidermaljunction with subepidermal blister formation. Specifically, autoreactiveB and T cell responses against the hemidesmosomal antigens BP180 andBP230 have been identified. Serum levels of autoantibodies to BP180reflect the disease severity and activity. The T cells are memory CD4+cells producing both Th1 and Th2 cytokines, mostly IL-4, IL-5 and IL-13.IL-5 as well as eotaxin are abundantly found in blister fluids. Theproduction of IL-5 is indeed associated with blood eosinophilia andsignificant eosinophil infiltration in the skin of BP patients.Eosinophils are thought to be critically implicated in blister formationby releasing toxic granule proteins (ESP, MBP) and proteolytic enzymes.

The term “eosinophilic esophagitis” (EoE) as used herein means anallergic inflammatory condition of the esophagus that involveseosinophils. Symptoms are swallowing difficulty, food impaction, andheartburn. EoE is characterised by a dense infiltrate with white bloodcells of the eosinophil type into the epithelial lining of theesophagus. EoE is believed to be an allergic reaction against ingestedfood, based on the important role eosinophils play in allergicreactions. The EoE diagnostic panel can be used to diagnose EoE. EoE canalso be diagnosed if gastroesophageal reflux does not respond to a 6week trial of twice-a-day high-dose proton-pump inhibitors (PPIs) or ifa negative ambulatory pH study ruled out gastroesophageal reflux disease(GERD). Endoscopically, ridges, furrows, or rings may be seen in theoesophageal wall. Sometimes, multiple rings may occur in the esophagus,leading to the term “corrugated esophagus” or “feline esophagus” due tosimilarity of the rings to the cat esophagus. The presence of whiteexudates in esophagus is also suggestive of the diagnosis. On biopsytaken at the time of endoscopy, numerous eosinophils can typically beseen in the superficial epithelium. A minimum of 15 eosinophils perhigh-power field are required to make the diagnosis. Eosinophilicinflammation is not limited to the oesophagus alone, and does extendthough the whole gastrointestinal tract. Profoundly degranulatedeosinophils may also be present, as may microabcesses and an expansionof the basal layer. Radiologically, the term “ringed esophagus” has beenused for the appearance of eosinophilic esophagitis on barium swallowstudies to contrast with the appearance of transient transverse foldssometimes seen with esophageal reflux (termed “feline esophagus”).

Treatment with compositions comprising mepolizumab may be used to treatCOPD according to the methods of the disclosure.

Subjects with “chronic obstructive pulmonary disease” (COPD) may meetone or more following criteria: a) a prior COPD diagnosis: subjects witha clinically documented history of COPD for at least 1 year inaccordance with the definition by the American Thoracic Society/EuropeanRespiratory Society; b) severity of COPD: Subjects may present with thefollowing: a measured pre and post-salbutamol Forced Expiratory Volumein one second/Forced vital capacity (FEV₁/FVC) ratio of <0.70 to confirma diagnosis of COPD; a measured post-salbutamol FEV₁>20 percent and <=80percent of predicted normal values calculated using National Health andNutrition Examination Survey (NHANES) III reference equations; c) ahistory of exacerbations: a well documented history (like medical recordverification) in the 12 months of: at least two moderate COPDexacerbations. Moderate is defined as the use of systemiccorticosteroids (IM, intravenous, or oral) and/or treatment withantibiotics, or at least one severe COPD exacerbation. Severe is definedas having required hospitalization. Note: At least one exacerbation musthave occurred while the subject was taking Inhaled corticosteroid (ICS)plus long acting beta2-agonist (LABA) plus long acting muscarinicantagonist (LAMA). Note: Prior use of antibiotics alone does not qualifyas a moderate exacerbation unless the use was specifically for thetreatment of worsening symptoms of COPD; and d) concomitant COPDtherapy: a well documented requirement for optimized standard of care(SoC) background therapy that includes ICS plus 2 additional COPDmedications (i.e., triple therapy) for the 12 months prior and meets thefollowing criteria: Immediately prior to visit to the healthcareprovider, a minimum of 3 months of use of an inhaled corticosteroid (ata dose>=500 micrograms (mcg)/day fluticasone propionate dose equivalentplus); or LABA and LAMA.

Treatment with compositions comprising mepolizumab may be used to treatCOPD according to the methods of the disclosure.

The term “eosinophilic granulomatosis with polyangiitis” (EGPA) as usedherein means an autoimmune condition that causes inflammation of smalland medium-sized blood vessels (vasculitis) in persons with a history ofairway allergic hypersensitivity (atopy). EGPA may also be referred toas Churg-Strauss Syndrome (CSS) or allergic granulomatosis. EGPA usuallymanifests in three stages. The early (prodromal) stage is marked byairway inflammation; almost all patients experience asthma and/orallergic rhinitis. The second stage is characterized by abnormally highnumbers of eosinophils (hypereosinophilia), which causes tissue damage,most commonly to the lungs and the digestive tract. The third stageconsists of vasculitis, which can eventually lead to cell death and canbe life-threatening.

Subjects with EGPA may meet one or more following criteria: a) asthma;b) blood eosinophil levels greater than 10% of a differential whiteblood cell count; c) presence of mononeuropathy or polyneuropathy; d)unfixed pulmonary infiltrates; e) presence of paranasal sinusabnormalities; and e) histological evidence of extravasculareosinophils. For classification purposes, a patient shall be said tohave EGPA if at least four of the preceding six criteria are positive.

Treatment with compositions comprising mepolizumab may be used to treatEGPA according to the methods of the disclosure. The compositions of thedisclosure may be administered to an EGPA patient in an amount of 300 mgonce every 4 weeks.

The term “hypereosinophilic syndrome” (HES) as used herein means adisease characterized by a persistently elevated eosinophil count (≥1500eosinophils/mm³) in the blood for at least six months without anyrecognizable cause, with involvement of either the heart, nervoussystem, or bone marrow.

Subjects with hypereosinophilic syndrome may meet one or more followingcriteria: a) a documented history of hypereosinophilic syndrome; b) ablood eosinophil count greater than 1500 cells for 6 months; c) signsand symptoms of organ system involvement; and d) no evidence ofparasitic, allergic or other causes of eosinophilia after comprehensiveevaluation.

Treatment with compositions comprising mepolizumab may be used to treathypereosinophilic syndrome according to the methods of the disclosure.The compositions of the disclosure may be administered to ahypereosinophilic syndrome patient in an amount of 300 mg once every 4weeks.

The term “nasal polyposis” as used herein means a disease characterizedby the presence of polyps nasal cavity. Such polyps may be in the uppernasal cavity and/or may originate from within the ostiomeatal complex.

Subjects with nasal polyposis may meet one or more following criteria:a) a documented history of nasal polyposis; or b) nasal polyps apparenton examination (e.g., endoscopic examination).

Treatment with compositions comprising mepolizumab may be used to treatnasal polyposis according to the methods of the disclosure. Thecompositions of the disclosure may be administered to a nasal polyposispatient in an amount of 750 mg once every 4 weeks.

The term “antibody” as used herein refers to molecules with animmunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD or IgE) andincludes monoclonal, recombinant, polyclonal, monoclonal, recombinant,polyclonal, chimeric, human, and humanized molecules of this type.Monoclonal antibodies may be produced by a eukaryotic cell cloneexpressing an antibody. Monoclonal antibodies may also be produced by aeukaryotic cell line which can recombinantly express the heavy chain andlight chain of the antibody by virtue of having nucleic acid sequencesencoding these introduced into the cell. Methods to produce antibodiesfrom different eukaryotic cell lines such as Chinese Hamster Ovarycells, hybridomas or immortalized antibody cells derived from an animal(e.g., human) are well known.

The antibody may be derived from rat, mouse, primate (e.g., cynomolgus,Old World monkey or Great Ape), human or other sources such as nucleicacids generated using molecular biology techniques which encode anantibody molecule.

The antibody may comprise a constant region, which may be of any isotypeor subclass. The constant region may be of the IgG isotype, for example,IgG₁, IgG₂, IgG₃, IgG₄ or variants thereof. The antigen binding proteinconstant region may be IgG₁.

The antigen binding protein may comprise one or more modificationsselected from a mutated constant domain such that the antibody hasenhanced effector functions/ADCC and/or complement activation.

An antibody may be capable of binding to a target antigen. Examples, ofsuch target antigens include human IL-5 comprising the amino acidsequence shown in SEQ ID NO: 11.

Mepolizumab comprising the heavy chain amino acid sequence shown in SEQID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2is an example of an antibody. Mepolizumab binds human IL-5 andantagonizes its activity.

Mepolizumab is a recombinant humanized monoclonal antibody (IgG₁, Kappa)Mepolizumab has two light and two heavy chains.

The mepolizumab heavy chain is encoded by the nucleic acid sequenceshown in SEQ ID NO: 13. The mepolizumab heavy chain contains 449 aminoacids with an estimated molecular mass of approximately 49 kDa. Thepredicted mature heavy chain amino acid sequence for mepolizumab is:

(SEQ ID NO: 1)

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIn the heavy chain amino acid sequence above, heavy chain frameworks andCDRs according to the Kabat definition are identified as zig-zagunderlined framework1, solid underlined CDR1, zig-zag underlinedframework2, solid underlined CDR2, zig-zag underlined framework3, solidunderlined CDR3 and zig-zag framework4 in order from the amino proximalportion to the carboxy terminal portion of the sequence presented. Inthe heavy chain amino acid sequence above, an asterisk to the right of acharacter for a single letter amino acid code indicates the amino acidresidue to the left can be a N-glycosylation site.

The mepolizumab light chain is encoded by the nucleic acid sequenceshown in SEQ ID NO: 14. The mepolizumab light chain contains 220 aminoacids residues with an estimated molecular mass of approximately 24 kDa.The mature light chain amino acid sequence is:

(SEQ ID NO: 2)

AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECIn the light chain amino acid sequence above, light chain frameworks andCDRs according to the Kabat definition are identified as zig-zagunderlined framework1, solid underlined CDR1, zig-zag underlinedframework2, solid underlined CDR2, zig-zag underlined framework3, solidunderlined CDR3 and zig-zag framework4 in order from the amino proximalportion to the carboxy terminal portion of the sequence presented.

The mepolizumab heavy and light chains are covalently linked by a singledisulfide bond and the heavy chains are linked to each other by twodisulfide bonds resulting in a typical IgG molecule. Both heavy chainscan be glycosylated at asparagine 299 with complex biantennaryoligosaccharides. The predicted polypeptide molecular mass is about 146kDa and the predicted carbohydrate molecular mass is approximately 3 kDagiving a total estimated molecular mass of 149.2 kDa for mepolizumab.Mepolizumab as encoded has 1338 amino acid residues (220 amino acidresidues per light chain, 449 amino acid residues per heavy chain). Themain pI of mepolizumab is about 8.7-9.1. The equilibrium dissociationconstant (K_(D)) for the molecular interaction of mepolizumab and humanIL-5 as measured using standard surface plasmon reasonance assays isless than 2.29×10⁻¹¹ M.

Mepolizumab can be provided as a lyophilized powder containing theantibody and excipients which can be reconstituted with apharmaceutically acceptable carrier (e.g., sterile water). Thisreconstituted pharmaceutical composition can then be administered eithersubcutaneously or intravenously (e.g., with further dilution).Mepolizumab can also be provided as a liquid formulation containing theantibody, excipients and a pharmaceutically acceptable carrier. Thisliquid pharmaceutical composition can then be administered eithersubcutaneously or intravenously (e.g., with further dilution).

The term “antibody variant” as used herein means an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification (e.g., by having a different amino acid side chain),post-translational modification or other modification in at least oneheavy chain, light chain, or combinations of these that results in astructural change (e.g., different amino acid side chain, differentpost-translational modification or other modification) relative to theparent antibody. Mepolizumab is an example of a such a parent antibody.Structural changes can be determined directly by a variety of methodswell know in the art such as LC-MS, direct sequencing or indirectly viamethods such as isoelectric focusing and the like. Such methods are wellknown to those of ordinary skill in the art.

The term “IL-5” as used herein means human IL-5 comprising the aminoacid sequence shown in SEQ ID NO: 11.

The term “specifically binds”, as used herein in relation to antigenbinding proteins means that the antigen binding protein binds to atarget antigen as well as a discrete domain, or discrete amino acidsequence, within a target antigen with no or insignificant binding toother (for example, unrelated) proteins. This term, however, does notexclude the fact that the antigen binding proteins may also becross-reactive with closely related molecules (for example, those with ahigh degree of sequence identity or from another genera or species). Theantigen binding proteins described herein may bind to human IL-5 or thehuman IL-5 receptor with at least 2, 5, 10, 50, 100, or 1000-foldgreater affinity than they bind to closely related molecules.

The binding affinity (K_(D)) of the antigen binding protein-targetantigen interaction may be 1 mM or less, 100 nM or less, 10 nM or less,2 nM or less or 1 nM or less. Alternatively, the K_(D) may be between 5and 10 nM; or between 1 and 2 nM. The K_(D) may be between 1 pM and 500pM; or between 500 pM and 1 nM. The binding affinity of the antigenbinding protein is determined by the association constant (Ka) and thedissociation constant (Kd) (KD=Kd/Ka). The binding affinity may bemeasured by BIACORE™, for example, by capture of the test antibody ontoa protein-A coated sensor surface and flowing target antigen over thissurface. Alternatively, the binding affinity can be measured byFORTEBIO, for example, with the test antibody receptor captured onto aprotein-A coated needle and flowing target antigen over this surface.

The K_(d) may be 1×10⁻³ Ms⁻¹ or less, 1×10⁻⁴ Ms⁻¹ or less, or 1×10⁻⁵Ms⁻¹ or less. The K_(d) may be between 1×10⁻⁵ Ms⁻¹ and 1×10⁻⁴ Ms⁻¹; orbetween 1×10⁻⁴ Ms⁻¹ and 1×10⁻³ Ms⁻¹. A slow K_(d) may result in a slowdissociation of the antigen binding protein-target antigen complex andimproved neutralization of the target antigen.

The term “specific antigen binding activity” as used herein meansantigen binding activity as measured by Surface Plasmon Resonance (SPR).IL-5 specific binding activity may be determined by SPR using a BIACORE™instrument, for example performed in the binding mode. It is bindingactivity divided by total protein (e.g., mepolizumab) content in asample.

The term “FcRn binding activity” as used herein means Neonatal Fc (FcRn)Receptor binding activity as measured by Surface Plasmon Resonance(SPR). FcRn binding may be determined using a BIACORE™ instrument. It isbinding activity to the FcRn receptor, divided by the total proteinconcentration of the sample.

The SPR method for specific antigen binding and FcRn binding uses areference standard of mepolizumab. The mepolizumab reference standardcan be used in assays to obtain system suitability and samplecomparability data, to ensure methods are performing appropriately. Thereference standard can allow the establishment of a calibration curveand concentrations of the samples are interpolated from the curve.

For example, the reference standard is a composition comprising SEQ IDNO:1 and SEQ ID NO:2. In another embodiment, the reference standard is acomposition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or more HCC-terminal lysine deleted variant, and 95% or more HC N-terminalpyro-glutamate variant. In a further embodiment, the reference standardis a composition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or moreHC C-terminal lysine deleted variant, 95% or more HC N-terminalpyro-glutamate variant, and 6% or less deamidated variant. In anotherembodiment, the reference standard is a composition comprising SEQ IDNO:1 and SEQ ID NO:2, and 98% or more HC C-terminal lysine deletedvariant, 95% or more HC N-terminal pyro-glutamate variant, 6% or lessdeamidated variant, 4% or less methionine or cysteine oxidated variant,and 0.1% tryptophan oxidated variant. In a further embodiment, thereference standard is a composition comprising SEQ ID NO:1 and SEQ IDNO:2, and 98% or more HC C-terminal lysine deleted variant, 95% or moreHC N-terminal pyro-glutamate variant, 6% or less deamidated variant, 4%or less methionine or cysteine oxidated variant, 0.1% or less tryptophanoxidated variant, and 0.4% or less aggregated variant. In anotherembodiment, the reference standard is a composition comprising theisoforms represented by peak 65, peak 78, peak 88, peak 92, the mainpeak and peak 112 shown in FIG. 1. In one embodiment the referencestandard is a composition comprising SEQ ID NO: 1 and SEQ ID NO:2, about62.9% main peak, about 35.9% acidic peak, about 1.2% basic peak, about99.6% monomer, about 0.4% aggregate, about 0% fragment, about 0.8% HCdeamidated N317, about 5.5% HC deamidated N386, about 5.2% HC deamidatedN31, about 0.2% HC deamidated N299, about 0.9% HC oxidised M64, about3.5% HC oxidised M254, about 0.5% HC oxidised M360, about 0.5% HCoxidised M430, about 0.3% HC oxidised M82 and M85, about 0.2% LCoxidised M4, about 0.0% LC oxidised C220, about 0.1% HC oxidised W52,98% or more HC C-terminal lysine deleted variant, and 95% or more HCN-terminal pyro-glutamate variant.

In one embodiment the composition has a specific IL-5 binding activityof ≥0.70; and a FcRn binding activity of ≥70%. For example, the specificantigen binding is in the range of from 0.70 to 1.30; and/or the FcRnbinding is in the range of from 70% to 130%, as compared to thereference standard which is set as 1.0 specific IL-5 binding activity,and 100% FcRn binding activity.

IL-5 neutralization ED₅₀ ratio is the ED₅₀ of a reference antibodystandard (e.g., a mepolizumab antibody standard comprising the aminoacid sequences of SEQ ID NO: 1 and SEQ ID NO: 2) divided by the ED₅₀ ofan antibody sample (e.g., a mepolizumab variant sample or a sample of amanufactured batch of composition comprising a mepolizumab antibodycomprising the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2).

By “isolated”, it is intended that the molecule, such as an antigenbinding protein or nucleic acid, is removed from the environment inwhich it may be found in nature. For example, the molecule may bepurified away from substances with which it would normally exist innature. For example, the mass of the molecule in a sample may be 95% ofthe total mass.

The terms “V_(H)” and “V_(L)” are used herein to refer to the heavychain variable region and light chain variable region respectively of anantigen binding protein.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antigen binding protein. These are the hypervariableregions of immunoglobulin heavy and light chains. There are three heavychain and three light chain CDRs (or CDR regions) in the variableportion of an immunoglobulin. Thus, “CDRs” as used herein refers to allthree heavy chain CDRs, all three light chain CDRs, all heavy and lightchain CDRs, or at least one CDR and wherein the at least one CDR isCDRH3. Framework regions follow each of these CDR regions. Acceptableheavy chain variable region and light chain variable region framework 1,framework 2 and framework 3 regions are readily recognized by those ofordinary skill in the art. Acceptable heavy chain constant regions(including hinge regions) and light chain constant regions are readilyrecognized by those of ordinary skill in the art as well. Acceptableantibody isotypes are similarly readily recognized by those of ordinaryskill in the art.

Throughout this specification, amino acid residues in variable domainsequences and full length antibody sequences are numbered according tothe Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”,“CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the specificationfollow the Kabat numbering convention.

It will be apparent to those skilled in the art that there arealternative numbering conventions for amino acid residues in variabledomain sequences and full length antibody sequences. There are alsoalternative numbering conventions for CDR sequences, for example thoseset out according to the Chothia numbering convention. The structure andprotein folding of the antibody may mean that other residues areconsidered part of the CDR sequence and would be understood to be so bya skilled person.

Other numbering conventions for CDR sequences available to a skilledperson include “AbM” (University of Bath) and “contact” (UniversityCollege London) methods. The minimum overlapping region using at leasttwo of the Kabat, Chothia, AbM and contact methods can be determined toprovide the “minimum binding unit”. The minimum binding unit may be asub-portion of a CDR.

Table 5 below represents one definition using each numbering conventionfor each CDR or binding unit. The Kabat numbering scheme is used inTable 5 to number the variable domain amino acid sequence. It should benoted that some of the CDR definitions may vary depending on theindividual publication used.

TABLE 5 Minimum Kabat CDR Chothia CDR AbM CDR Contact CDR binding unitH1 31-35/35A/35B 26-32/33/34 26-35/35A/35B 30-35/35A/35B 31-32 H2 50-6552-56 50-58 47-58 52-56 H3  95-102  95-102  95-102  93-101  95-101 L124-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L3 89-9789-97 89-97 89-96 89-96

“Percent identity” between a query nucleic acid sequence and a subjectnucleic acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTN algorithm when a subjectnucleic acid sequence has 100% query coverage with a query nucleic acidsequence after a pair-wise BLASTN alignment is performed. Such pair-wiseBLASTN alignments between a query nucleic acid sequence and a subjectnucleic acid sequence are performed by using the default settings of theBLASTN algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query sequence may be described by a nucleic acidsequence identified in one or more claims herein.

Nucleic acid sequences which may be useful, and included, in thecompositions and related methods of the disclosure may have betweenabout 85% to about 100%, about 90% to about 100%, about 95% to about100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% and about 100% identity to the nucleicacid sequences identified in the disclosure (e.g., nucleic acidsencoding an antibody heavy chain or antibody light chain). In thedisclosure, percent identity between the nucleic acid sequencesdescribed may include any discrete subrange of the percent identiyranges recited above (e.g., any range of integer values within aparticular range or discrete subvalues within a particular range).

“Percent identity” between a query amino acid sequence and a subjectamino acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTP algorithm when a subjectamino acid sequence has 100% query coverage with a query amino acidsequence after a pair-wise BLASTP alignment is performed. Such pair-wiseBLASTP alignments between a query amino acid sequence and a subjectamino acid sequence are performed by using the default settings of theBLASTP algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query sequence may be described by an amino acidsequence identified in one or more claims herein.

The amino acid sequences which may be useful, and included, incompositions and related methods of the disclosure may have betweenabout 85% to about 100%, about 90% to about 100%, about 95% to about100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% and about 100% identity to the aminoacid sequences identified in the disclosure (e.g., to an antibody heavychain or antibody light chain). In the disclosure, percent identitybetween the amino acid sequences described may includes any discretesubrange of the percent identity ranges recited above (e.g., any rangeof integer values within a particular range or discrete subvalues withina particular range).

The terms “peptide”, “polypeptide”, “protein” and “peptide chain” eachrefer to a molecule comprising two or more amino acid residues. Apeptide may be monomeric or polymeric.

It is well recognized in the art that certain amino acid substitutionsare regarded as being “conservative” Amino acids are divided into groupsbased on common side-chain properties and substitutions within groupsthat maintain all or substantially all of the binding affinity of theantigen binding protein are regarded as conservative substitutions. SeeTable 6. The antigen binding proteins disclosed herein can comprise such“conservative” amino acid substitutions.

TABLE 6 Side chain Members Hydrophobic met, ala, val, leu, ile Neutralhydrophilic cys, ser, thr Acidic asp, glu Basic asn, gln, his, lys, argResidues that influence chain orientation gly, pro Aromatic trp, tyr,phe

The term “pharmaceutical compostion” as used herein means a compositionsuitable for administration to a patient.

The pharmaceutical compositions described herein may comprise purifiedpreparations of an antibody as described herein.

For example, the pharmaceutical preparation may comprise a purifiedpreparation of an antibody as described herein in combination with apharmaceutically acceptable carrier.

Typically, such pharmaceutical compositions comprise a pharmaceuticallyacceptable carrier as known and called for by acceptable pharmaceuticalpractice. Examples of such carriers include sterilized carriers, such assaline, Ringers solution, or dextrose solution, optionally buffered withsuitable buffers to a pH within a range of 5 to 8.

Pharmaceutical compositions may be administered by injection or infusion(e.g., intravenous, intraperitoneal, intradermal, subcutaneous,intramuscular, or intraportal). Such compositions are suitably free ofvisible particulate matter. Pharmaceutical compositions may comprisebetween 1 mg to 10 g of antigen binding protein, for example, between 5mg and 1 g of antigen binding protein. Alternatively, the compositionmay comprise between 5 mg and 500 mg of antigen binding protein, forexample, between 5 mg and 50 mg.

Methods for the preparation of such pharmaceutical compositions are wellknown to those skilled in the art. Pharmaceutical compositions maycomprise between 1 mg to 10 g of antigen binding protein in unit dosageform, optionally together with instructions for use. Pharmaceuticalcompositions may be lyophilized (freeze dried) for reconstitution priorto administration according to methods well known or apparent to thoseskilled in the art. Where antibodies have an IgG₁ isotype, a chelator ofcopper, such as citrate (e.g., sodium citrate) or EDTA or histidine, maybe added to the pharmaceutical composition to reduce the degree ofcopper-mediated degradation of antibodies of this isotype.Pharmaceutical compositions may also comprise a solubilizer, such asarginine, a surfactant/anti-aggregation agent such as polysorbate 80,and an inert gas such as nitrogen to replace vial headspace oxygen.

The term “therapeutically effective amount” as used herein means anamount of an agent (such as an antibody or a pharmaceuticalcomposition), which provides a therapeutic benefit in the treatment ormanagement of one or more symptoms of a condition to be treated (such asasthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma,eosinophilic asthma, sub-eosinophilic asthma, chronic obstructivepulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA),hypereosinophilic syndrome and nasal polyposis). Examples of suchtreatment or management of one or more symptoms of asthma—includingsevere eosinophilic asthma, uncontrolled eosinophilic asthma,eosinophilic asthma or sub-eosinophilic asthma—include 1) a reduction ofthe frequency of asthma exacerabations; 2) a reduction in the time tofirst clinically significant exacerbation requiring oral or systemiccorticosteroids, hospitalisation, and/or emergency department (ED)visits; 3) a reduction in the frequency of exacerbations requiringhospitalization (including intubation and admittance to an intensivecare unit) or ED visits; 4) a reduction in the time to firstexacerbation requiring hospitalization or ED visit; 5) a change frombaseline in clinic pre-bronchodilator FEV₁; 6) a change from baseline inclinic post-bronchodilator FEV₁; 7) a change from baseline in an AsthmaControl Questionnaire (ACQ) score; 8) improved lung function as assessedby spirometry (e.g., vital capacity (VC), forced vital capacity (FVC),forced expiratory volume (FEV) at timed intervals of 0.5, 1.0 (FEV₁),2.0, and 3.0 seconds, forced expiratory flow 25-75% (FEF 25-75) andmaximal voluntary ventilation (MVV) total lung capacity, idal volume,residual volume, expiratory reserve volume, inspiratory reserve volume,inspiratory capacity, inspiratory vital capacity, vital capacity,functional residual capacity, residual volume expressed as percent oftotal lung capacity, alveolar gas volume, actual volume of the lungincluding the volume of the conducting airway, forced vital capacity,etc.); and 9) a reduction in asthma exacerbations requiring steroids forcontrol (such as oral steroids or steroids—like prednisone, prednisoloneetc.—administered by any route). Such a reduction in asthmaexacerbations requiring steroids for control may be an approximately 50%reduction in exacerbations requiring steroids (e.g., oral steroids).

Therapeutically effective amounts and treatment regimes are generallydetermined empirically and may be dependent on factors, such as the age,weight, and health status of the patient and disease or disorder to betreated. Such factors are within the purview of the attending physician.

The dosage of antigen binding protein administered to a subject isgenerally between 1 μg/kg to 150 mg/kg, between 0.1 mg/kg and 100 mg/kg,between 0.5 mg/kg and 50 mg/kg, between 1 and 25 mg/kg, between about0.3 mg/kg and about 3 mg/kg or between 1 and 10 mg/kg of the subject'sbody weight. For example, the dose may be 10 mg/kg, 30 mg/kg, or 60mg/kg. The dose may also be from 10 mg/kg to 110 mg/mg 15 mg/kg to 25mg/kg or 15 mg/kg to 100 mg/kg. The antigen binding protein may beadministered, for example, parenterally, subcutaneously, intravenously,or intramuscularly. Doses may also be administered on a per subjectbasis such as about 20 mg per subject to about 750 mg per subject, about75 mg per subject to about 750 mg per subject, about 20 mg per subjectto about 200 mg per subject. The dose may be any discrete subrange withthese dosage ranges. For example, the dose (such as a dose ofmepolizumab or a pharmaceutical composition comprising mepolizumab) mayalso be administered subcutaneously on a per subject basis such as about100 mg per subject (e.g., once every four weeks), or 300 mg per subject(or other doses administered may be subcutaneously with providedapproximately the same, or comparable, bioavailability is achieved aswith intravenous administration—e.g., three doses of 100 mg per subjectto achieve a total dose administered subcutaneously of 300 mg persubject).

Ranges provided herein, of any type, include all values within aparticular range described and values about an endpoint for a particularrange.

If desired, the effective daily dose of a therapeutic composition may beadministered as two, three, four, five, six or more doses administeredseparately at appropriate intervals throughout the day, optionally, inunit dosage forms.

The administration of a dose may be by slow continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours, or from 2to 6 hours. Such an administration may result in reduced side effects.

The administration of a dose may be repeated one or more times asnecessary, for example, three times daily, once every day, once every 2days, once a week, once a every 14 days, once a month, once every 3months, once every 4 months, once every 6 months, or once every 12months. The antigen binding proteins may be administered by maintenancetherapy, for example once a week for a period of 6 months or more. Theantigen binding proteins may be administered by intermittent therapy,for example, for a period of 3 to 6 months and then no dose for 3 to 6months, followed by administration of antigen binding proteins again for3 to 6 months, and so on, in a cycle.

For example, the dose may be administered subcutaneously, once every 14or 28 days, in the form of multiple doses on each day of administration.In one embodiment, the dosage of the composition is 100 mg once every 4weeks (28 days).

The antigen binding protein may be administered to the subject in such away as to target therapy to a particular site.

The antigen binding protein in the methods of the disclosure may be usedin combination with one or more other therapeutically active agents,such as antibodies or small molecule inhibitors

By the term “treating” and grammatical variations thereof as usedherein, is meant therapeutic therapy. In reference to a particularcondition, treating means: (1) to ameliorate the condition of one ormore of the biological manifestations of the condition, (2) to interferewith a) one or more points in the biological cascade that leads to or isresponsible for the condition or b) one or more of the biologicalmanifestations of the condition, (3) to alleviate one or more of thesymptoms, effects or side effects associated with the condition ortreatment thereof, (4) to slow the progression of the condition or oneor more of the biological manifestations of the condition or (5) toprevent the onset of one or more of the biological manifistations of thecondition. Prophylactic therapy is also contemplated thereby. Theskilled artisan will appreciate that “prevention” is not an absoluteterm. In medicine, “prevention” is understood to refer to theprophylactic administration of a drug to substantially diminish thelikelihood or severity of a condition or biological manifestationthereof, or to delay the onset of such condition or biologicalmanifestation thereof.

The terms “individual”, “subject” and “patient” are used hereininterchangeably. The subject is typically a human. The subject may alsobe a mammal, such as a mouse, rat, or primate (e.g., a marmoset ormonkey). The subject can be a non-human animal. The antigen bindingproteins, compositions and methods of the disclosure also haveveterinary use. The subject to be treated may be a farm animal, forexample, a cow or bull, sheep, pig, ox, goat or horse, or may be adomestic animal such as a dog or cat. The animal may be any age, or amature adult animal.

Treatment can be therapeutic, prophylactic or preventative. The subjectwill be one who is in need thereof. Those in need of treatment mayinclude individuals already suffering from a particular medical disease,in addition to those who may develop the disease in the future.

Thus, the methods, antigen binding proteins and compositions of thedisclosure described herein can be used for prophylactic treatment orpreventative treatment if specified. In this case, methods, antigenbinding proteins and compositions of the disclosure can be used toprevent or delay the onset of one or more aspects or symptoms of adisease. The subject can be asymptomatic. The subject may have a geneticpredisposition to the disease. A prophylactically effective amount ofthe antigen binding protein is administered to such an individual. Aprophylactically effective amount is an amount which prevents or delaysthe onset of one or more aspects or symptoms of a disease describedherein.

The methods, antigen binding proteins and compositions of the disclosureneed not affect a complete cure, or eradicate every symptom ormanifestation of the disease to constitute a viable therapeutictreatment. As is recognised in the art, drugs employed as therapeuticagents in methods of treatment may reduce the severity of a givendisease state, but need not abolish every manifestation of the diseaseto be regarded as useful therapeutic agents. Similarly, aprophylactically administered treatment need not be completely effectivein preventing the onset of a disease in order to constitute a viableprophylactic agent. Simply reducing the impact of a disease (forexample, by reducing the number or severity of its symptoms, or byincreasing the effectiveness of another treatment, or by producinganother beneficial effect), or reducing the likelihood that the diseasewill occur (for example by delaying the onset of the disease) or worsenin a subject, is sufficient.

One aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤80% acidic antibody variants.

In one embodiment the composition has: a) ≥0.70 IL-5 specific antigenbinding; and/or b) ≥70% FcRn binding. IL-5 specific antigen binding,such as binding to human IL-5 comprising the amino acid sequence of SEQID NO: 11, can be measured using standard assays, such as surfaceplasmon resonance (e.g., BIACORE™), that are well known in the art. FcRnbinding can similarly be measured using standard assays, such as surfaceplasmon resonance (e.g., BIACORE™), that are well known in the art.

In another embodiment a) the specific antigen binding is in the range offrom 0.70 to 1.30; and/or b) the FcRn binding is in the range of from70% to 130%. In some embodiments the specific antigen binding may be inthe range of about 0.9 to 1.1, 0.75 to about 1, about 0.7 to about 0.8,about 0.7, about 0.91 to about 0.95, about 0.994 to to about 0.997 orabout 0.7 to about 0.9. In some embodiments the FcRn binding is in therange of from about 70% to about 100%, about 100% to about 130%, about70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 80%to about 90%, about 80% to about 100%, about 100% to about 110%, about110% to about 120%, about 120% to about 130%, about 80% to about 120%and about 90% to about 110%.

In one embodiment, the composition comprises: ≤80% acidic antibodyvariants. For example, the composition may comprise ≤75%, ≤70%, ≤65%,≤60%, ≤55%, ≤50%, or ≤45% acidic antibody variants.

In another embodiment the composition comprises: ≤35% deamidatedantibody variants.

In another embodiment the composition comprises: ≤25% deamidatedantibody variants at N31 of the light chain amino acid sequence. Forexample, the composition may comprise ≤22.5%, ≤20%, ≤17.5%, ≤15%, ≤12.5,≤10%, or ≤7.5% deamidated antibody variants at N31 of the light chainamino acid sequence.

In another embodiment the composition comprises: ≤35% deamidatedantibody variants at N386 of the heavy chain amino acid sequence. Forexample, the composition may comprise ≤32.5%, ≤30%, ≤25.5%, or ≤20%,≤17.5%, ≤15%, ≤12.5%, ≤10%, or ≤7.5% deamidated antibody variants atN386 of the heavy chain amino acid sequence.

In another embodiment the composition comprises: ≤55% oxidised antibodyvariants.

In another embodiment the composition comprises: ≤55% oxidised antibodyvariant at any one or a combination of: a) M64 of the heavy chain aminoacid sequence; b) M254 of the heavy chain amino acid sequence; and/or c)M430 of the heavy chain amino acid sequence. For example, thecomposition may comprise ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, or ≤25%, ≤20%,≤15%, ≤10%, or ≤5% oxidised antibody variants at M64, M254, and/or M430of the heavy chain amino acid sequence.

In another embodiment the composition comprises: ≤3% oxidised antibodyvariants at W52 of the heavy chain amino acid sequence. For example, thecomposition may comprise ≤2.5%, ≤2%, ≤1.5%, ≤1%, ≤0.5%, ≤0.4%, ≤0.3%,≤0.25%, ≤0.2%, ≤0.15%, or ≤0.1% oxidised antibody variants at W52 of theheavy chain amino acid sequence.

In another embodiment a deamidated antibody variant amount and/or anoxidised variant amount, is determined by peptide mapping LC-MS/MS.

In another embodiment the composition comprises: ≤20% aggregatedantibody variants. For example, the composition may comprise ≤17.5%,≤15%, ≤12.5, ≤10%, ≤7.5%, ≤5%, or ≤4%, aggregated variant. Thecompositions may comprise less than or equal to 3%, 2%, 1% or 0.5%aggregated antibody. The composition may comprise greater than or equalto 98% monomeric antibody.

In another embodiment the aggregated antibody variant comprises a dimer.Such an aggregated antibody can comprise two antibody molecules (e.g.,two IgG1 antibody molecules).

In another embodiment the aggregated antibody variant amount isdetermined by size exclusion chromatography (SEC). Methods forperforming size exclusion chromatography and measuring protein moleculesize are well known in the art.

In another embodiment the composition comprises: ≥50% heavy chain aminoacid sequence C-terminal lysine K449 deleted antibody variants. Forexample, the composition may comprise ≥60%, ≥70%, ≥75, ≥80%, ≥85%, ≥90%,or ≥95% heavy chain amino acid sequence C-terminal lysine K449 deletedantibody variants.

In another embodiment the composition comprises: ≥50% heavy chain aminoacid sequence pyro-glutamate N-terminal antibody variants. For example,the composition may comprise ≥60%, ≥70%, ≥75, ≥80%, ≥85%, ≥90%, or ≥95%heavy chain amino acid sequence pyro-glutamate N-terminal antibodyvariants.

In another embodiment the composition comprises Host Cell Protein (HCP).The HCP may be CHO cell derived. HCP is a process-related impurity incontrast to mepolizumab product-related substances (i.e. mepolizumabplus mepolizumab variants). Industry standard acceptable limits for HCPcan be up to 100 ppm (equal to 100 ng/mg). HCP content in a compositiondescribed herein may be ≤50 ng/mg, ≤40 ng/mg, ≤30 ng/mg, or ≤20 ng/mg.For example, HCP content of the composition may be ≤10 ng/mg. In aparticular embodiment, HCP content of the composition may be ≤5 ng/mg or≤2 ng/mg.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤80% acidic antibody variants and≤20% aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; and ≤20% aggregated antibodyvariants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤55% oxidised antibodyvariants at M64 of the heavy chain amino acid sequence; ≤3% oxidisedvariants at W52 of the heavy chain amino acid sequence; and ≤20%aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤35% deamidated antibodyvariants at N386 of the heavy chain amino acid sequence; and ≤20%aggregated antibody variants.

Another aspect of the disclosure is a composition comprising an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2, or an antibodyvariant having a heavy chain amino acid sequence at least 90% identicalto the heavy chain amino acid sequence and/or a light chain amino acidsequence at least 90% identical to the light chain amino acid sequence,wherein the composition comprises: ≤25% deamidated antibody variants atN31 of the light chain amino acid sequence; ≤35% deamidated antibodyvariants at N386 of the heavy chain amino acid sequence; ≤55% oxidisedantibody variants at M64 of the heavy chain amino acid sequence, M254 ofthe heavy chain amino acid sequence, M430 of the heavy chain amino acidsequence; ≤3% oxidised antibody variants at W52 of the heavy chain aminoacid sequence; and ≤20% aggregated antibody variants.

The compositions of the disclosure may further comprise a bufferingagent selected from the group consisting of sodium phosphate dibasicheptahydrate, phosphate, citric acid, citrate, sodium phosphate,potassium phosphate, sodium citrate, and histidine, providing a pH ofbetween 6.8 and 7.2 or a pH of from pH 6.2 to pH 6.6 with a pH value of6.3 being preferred. The buffer in the compositions of the disclosuremay be present in the range from about 10-30 mM, about 10-20 mM, about20 mM or about 15.5 mM. For example, the buffer in the compositions ofthe disclosure is present at about 20 mM, or at about 15.5 mM sodiumphosphate dibasic heptahydrate.

The compositions of the disclosure may comprise sodium phosphate dibasicheptahydrate and citric acid buffering agents providing a pH of from 6.2to 6.6 inclusive with a pH value of 6.3 being preferred. The sodiumphosphate dibasic heptahydrate buffering agent may be present in therange from about 15-16.4 mM and the citric acid buffering agent may bepresent in the range from about 3.8-4.9 mM. For example, thecompositions of the disclosure may comprise about 15.5 mM sodiumphosphate dibasic heptahydrate and about 4.5 mM citric acid monohydrate.

The compositions of the disclosure may further comprise a sugar. Thecompositions of the disclosure may further comprise sucrose. Sucrose maybe present in the compositions of the disclosure in the range from about5-20%; about 10-15%, about 11-13% or at about 12% weight by volume.

The compositions of the disclosure may further comprise polysorbate 80.Polysorbate 80 may be present in the range from about 0.01-0.1% weightby volume. For example, polysorbate 80 may be present in thecompositions of the disclosure at about 0.02% weight by volume, or atabout 0.05% weight by volume.

The compositions of the disclosure may further comprise EDTA. EDTA maybe present in the range from about 0.01-0.1 mM. For example, EDTA may bepresent at about 0.05 mM.

In one embodiment, the compositions of the disclosure further comprise20 mM sodium phosphate dibasic heptahydrate, 12% weight of sucrose tovolume and 0.05% weight of polysorbate 80 to volume.

In another embodiment, the compositions of the disclosure furthercomprise 15.5 mM sodium phosphate dibasic, 3.9 mM citric acidmonohydrate, 12% weight of sucrose to volume, 0.02% weight ofpolysorbate 80 to volume and 0.05 mM EDTA.

The compositions of the disclosure may comprise an aqueous liquidformulation at pH 6.2 containing 16.1 mM sodium phosphate dibasicheptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.

The compositions of the disclosure may comprise an aqueous liquidformulation at pH 6.2 containing 15.2 mM sodium phosphate dibasicheptahydrate, 4.8 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.

The compositions of the disclosure may comprise an aqueous liquidformulation at pH 6.4 containing 15.8 mM sodium phosphate dibasicheptahydrate, 4.2 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.

The compositions of the disclosure may comprise an aqueous liquidformulation at pH 6.6 containing 16.3 mM sodium phosphate dibasicheptahydrate, 3.7 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.

The compositions of the disclosure may comprise an aqueous liquidformulation at pH 6.3 containing 15.5 mM sodium phosphate dibasicheptahydrate, 4.5 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.Importantly, the tangential filtration and ultrafiltration exchange stepof Example 1 below may be adjusted to produce the compositions of thedisclosure, such as a composition of the disclosure comprising 15.5 mMsodium phosphate dibasic heptahydrate, 4.5 mM citric citric acidmonohydrate, 12% weight to volume sucrose, 0.02% weight to volumepolysorbate 80, 0.05 mM EDTA at a pH of 6.3—or other such liquidformulations.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.8 to 7.2, wherein the buffering agent ishistidine, phosphate, citric acid, citrate or a salt thereof, whereinthe purified preparation comprises the isoforms represented by peak 65,peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1,wherein the antibody comprises a heavy chain amino acid sequence havingat least 90% identity to the amino acid sequence of SEQ ID NO: 1 and alight chain amino acid sequence having at least 90% identity to theamino acid sequence of SEQ ID NO: 2, and wherein the antibody isproduced by a Chinese Hamster Ovary cell. In the composition the heavychain may comprise an amino acid sequence having at least 95%, 96%,96.88%, 97%, 98% or 99% identity to the amino acid sequence of SEQ IDNO: 1. In the composition the light chain may comprise an amino acidsequence having at least 98%, 98.63 or 99% identity to the amino acidsequence of SEQ ID NO: 2.

In one embodiment the buffering agent is at least one selected from thegroup consisting of sodium phosphate dibasic heptahydrate, phosphate,citric acid and citrate.

In another embodiment the buffering agent is sodium phosphate, potassiumphosphate, or sodium citrate.

In another embodiment the composition further comprises a sugar, acarbohydrate and/or a salt.

In another embodiment the composition comprises sucrose.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.8 to 7.2, wherein the buffering agent isphosphate or a salt thereof, wherein the purified preparation comprisesthe isoforms represented by peak 65, peak 78, peak 88, peak 92, the mainpeak and peak 112 shown in FIG. 1, wherein the antibody comprises aheavy chain amino acid sequence having at least 90% identity to theamino acid sequence of SEQ ID NO: 1 and a light chain amino acidsequence having at least 90% identity to the amino acid sequence of SEQID NO: 2, and wherein the antibody is produced by a Chinese HamsterOvary cell.

In another embodiment the buffering agent is at least one selected fromthe group consisting of sodium phosphate dibasic heptahydrate,phosphate, citric acid and citrate.

In another embodiment the composition further comprises a sugar.

In another embodiment the sugar is sucrose.

In another embodiment the composition comprises polysorbate 80.

In another embodiment the composition comprises one selected from afirst formulation of 20 mM sodium phosphate dibasic heptahydrate, 12%weight of sucrose to volume and 0.05% weight of polysorbate 80 tovolume; and a second formulation of 15.5 mM sodium phosphate dibasicheptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose tovolume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; and athird formulation of 26 mM sodium phosphate dibasic heptahydrate, 15%weight of sucrose to volume and 0.065% weight of polysorbate 80 tovolume. The composition may be at a pH between about 6.8 to about 7.2,about 6.1 to about 6.5 or about 6 to about 6.6.

In another embodiment the antibody has a dissociation constant equal to,or less than, about 3.5×10⁻¹¹ M for human interleukin-5 comprising theamino acid sequence shown in SEQ ID NO: 11.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) a main form of the antibody comprisinggreater than, or equal to, 50% of the protein in the composition asmeasured using capillary isoelectric focusing of the composition. Themain form of the antibody may also comprise greater than, or equal to,57.9%, 59.4%, and 60% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition.

In another embodiment the main form of the antibody comprises at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) acidicforms of the antibody comprising about 20% to about 45% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition. The acid forms of the antibody may also comprisegreater than, or equal to, 37.6%, 37.8%, 38.4% and 39.8% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition. The total acidic peak area determined by cIEF can be ashigh as 72% and still retain 0.74 IL-5 specific binding and 80% FcRnbinding.

In another embodiment the acidic forms of the antibody comprise at leastone selected from the group consisting of a peak 65 acidic form, a peak78 acidic form, a peak 88 acidic form and a peak 92 acidic form.

In another embodiment the acidic forms of the antibody comprise at leastone deamidated amino acid residue selected from the group consisting ofa heavy chain amino acid residue deamidated at asparagine 299, a heavychain amino acid residue deamidated at asparagine 317, a heavy chainamino acid residue deamidated at asparagine 386 and a light chain aminoacid residue deamidated at asparagine 31. An acceptable level ofdeamidation on LC N31 is greater than or equal to 17%, or greater thanor equal to 17.4% as measured by peptide mapping LC MS/MS). Anacceptable level of deamidation on HC 386 is greater than or equal to30% as measured by peptide mapping LC MS/MS). The acceptable upper levelmay be the level of a particular variant that allows the antibodymolecules in the composition to retain antigen binding activity of about0.70 to about 1.30 as measured by SPR and FcRn binding activity of about70% to about 130% as measured by SPR or other antigen binding activityor FcRn binding activity values, or ranges, disclosed herein.

In another embodiment the acidic forms of the antibody comprise at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220. Acceptable levels of oxidation on theheavy chain residues of the antibody as measured by peptide mappingLC-MS/MS may be about 50% for HC M64, M254, and M430 and about 3% for HCW52. The acceptable upper level may be the level of a particular variantthat allows the antibody molecules in the composition to retain antigenbinding activity of about 0.70 to about 1.30 as measured by SPR and FcRnbinding activity of about 70% to about 130% as measured by SPR or otherantigen binding activity or FcRn binding activity values, or ranges,disclosed herein.

In another embodiment the main form of the antibody comprises at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220; and the acidic forms of the antibodycomprise at least one oxidized amino acid residue selected from thegroup consisting of a heavy chain amino acid residue oxidized attryptophan 52, a heavy chain amino acid residue oxidized at methionine64, a heavy chain amino acid residue oxidized at methionine 82, a heavychain amino acid residue oxidized at methionine 85, a heavy chain aminoacid residue oxidized at cysteine 222, a heavy chain amino acid residueoxidized at methionine 254, a heavy chain amino acid residue oxidized atmethionine 360, a heavy chain amino acid residue oxidized at methionine430, a light chain amino acid residue oxidized at methionine 4 and alight chain amino acid residue oxidized at cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) a basicform of the antibody comprising about 1% to about 15% of the protein inthe composition as measured using capillary isoelectric focusing of thecomposition. The basic form of the antibody may also comprise greaterthan, or equal to, 2.2% and 2.3% of the protein in the composition asmeasured using capillary isoelectric focusing of the composition.

In another embodiment the basic form of the antibody comprises a peak112 basic form.

In another embodiment the basic form of the antibody comprises a heavychain having a carboxy terminal residue that is glycine 448.

In another embodiment the basic forms of the antibody comprise at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220.

In another embodiment the main form of the antibody comprises at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220; and the basic forms of the antibodycomprise at least one oxidized amino acid residue selected from thegroup consisting of a heavy chain amino acid residue oxidized attryptophan 52, a heavy chain amino acid residue oxidized at methionine64, a heavy chain amino acid residue oxidized at methionine 82, a heavychain amino acid residue oxidized at methionine 85, a heavy chain aminoacid residue oxidized at cysteine 222, a heavy chain amino acid residueoxidized at methionine 254, a heavy chain amino acid residue oxidized atmethionine 360, a heavy chain amino acid residue oxidized at methionine430, a light chain amino acid residue oxidized at methionine 4 and aheavy chain amino acid residue oxidized at cysteine 222.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 50% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; c) acidic formsof the antibody comprising about 20% to about 45% of the protein in thecomposition as measured using capillary isoelectric focusing of thecomposition; and d) a basic form of the antibody comprising about 1% toabout 15% of the protein in the composition as measured using capillaryisoelectric focusing of the composition.

In another embodiment the acidic forms of the antibody comprise at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220; and the basic forms of the antibodycomprise at least one oxidized amino acid residue selected from thegroup consisting of a heavy chain amino acid residue oxidized attryptophan 52, a heavy chain amino acid residue oxidized at methionine64, a heavy chain amino acid residue oxidized at methionine 82, a heavychain amino acid residue oxidized at methionine 85, a heavy chain aminoacid residue oxidized at cysteine 222, a heavy chain amino acid residueoxidized at methionine 254, a heavy chain amino acid residue oxidized atmethionine 360, a heavy chain amino acid residue oxidized at methionine430, a light chain amino acid residue oxidized at methionine 4 and aheavy chain amino acid residue oxidized at cysteine 222.

In another embodiment the main form of the antibody comprises at leastone oxidized amino acid residue selected from the group consisting of aheavy chain amino acid residue oxidized at tryptophan 52, a heavy chainamino acid residue oxidized at methionine 64, a heavy chain amino acidresidue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85, a heavy chain amino acid residue oxidized atcysteine 222, a heavy chain amino acid residue oxidized at methionine254, a heavy chain amino acid residue oxidized at methionine 360, aheavy chain amino acid residue oxidized at methionine 430, a light chainamino acid residue oxidized at methionine 4 and a light chain amino acidresidue oxidized at cysteine 220; the acidic forms of the antibodycomprise at least one oxidized amino acid residue selected from thegroup consisting of a heavy chain amino acid residue oxidized attryptophan 52, a heavy chain amino acid residue oxidized at methionine64, a heavy chain amino acid residue oxidized at methionine 82, a heavychain amino acid residue oxidized at methionine 85, a heavy chain aminoacid residue oxidized at cysteine 222, a heavy chain amino acid residueoxidized at methionine 254, a heavy chain amino acid residue oxidized atmethionine 360, a heavy chain amino acid residue oxidized at methionine430, a light chain amino acid residue oxidized at methionine 4 and alight chain amino acid residue oxidized at cysteine 220; and wherein thebasic forms of the antibody comprise at least one oxidized amino acidresidue selected from the group consisting of a heavy chain amino acidresidue oxidized at tryptophan 52, a heavy chain amino acid residueoxidized at methionine 64, a heavy chain amino acid residue oxidized atmethionine 82, a heavy chain amino acid residue oxidized at methionine85, a heavy chain amino acid residue oxidized at cysteine 222, a heavychain amino acid residue oxidized at methionine 254, a heavy chain aminoacid residue oxidized at methionine 360, a heavy chain amino acidresidue oxidized at methionine 430, a light chain amino acid residueoxidized at methionine 4 and a light chain amino acid residue oxidizedat cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) deamidated forms of the antibodycomprising at least one selected from the group consisting of a heavychain amino acid residue deamidated at asparagine 299, a heavy chainamino acid residue deamidated at asparagine 317, a heavy chain aminoacid residue deamidated at asparagine 386 and a light chain amino acidresidue deamidated at asparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) oxidized forms of the antibody comprisingat least one selected from the group consisting of a heavy chain aminoacid residue oxidized at tryptophan 52, a heavy chain amino acid residueoxidized at methionine 64, a heavy chain amino acid residue oxidized atmethionine 82, a heavy chain amino acid residue oxidized at methionine85, a heavy chain amino acid residue oxidized at cysteine 222, a heavychain amino acid residue oxidized at methionine 254, a heavy chain aminoacid residue oxidized at methionine 360, a heavy chain amino acidresidue oxidized at methionine 430, a light chain amino acid residueoxidized at methionine 4 and a light chain amino acid residue oxidizedat cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) deamidated forms of the antibody comprising atleast one selected from the group consisting of a heavy chain amino acidresidue deamidated at asparagine 299, a heavy chain amino acid residuedeamidated at asparagine 317, a heavy chain amino acid residuedeamidated at asparagine 386 and a light chain amino acid residuedeamidated at asparagine 31; and c) oxidized forms of the antibodycomprising at least one selected from the group consisting of a heavychain amino acid residue oxidized at tryptophan 52, a heavy chain aminoacid residue oxidized at methionine 64, a heavy chain amino acid residueoxidized at methionine 82, a heavy chain amino acid residue oxidized atmethionine 85, a heavy chain amino acid residue oxidized at cysteine222, a heavy chain amino acid residue oxidized at methionine 254, aheavy chain amino acid residue oxidized at methionine 360, a heavy chainamino acid residue oxidized at methionine 430, a light chain amino acidresidue oxidized at methionine 4 and a light chain amino acid residueoxidized at cysteine 220.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31. In the composition CDRH2may comprise an amino acid sequence having at least 85% or 87.5%identity to the amino acid sequence of SEQ ID NO: 6. In the compositionCDRL1 may comprise an amino acid sequence having at 93%, 94% or 94.11%identity to the amino acid sequence of SEQ ID NO: 8.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) oxidized forms of the antibody comprising a heavy chainamino acid residue oxidized at methionine 64.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region having theCDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acidsequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shownin SEQ ID NO: 7; and a light chain variable region having the CDRL1amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequenceshown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ IDNO: 10; and b) oxidized forms of the antibody comprising a heavy chainamino acid residue oxidized at methionine 64; and c) deamidated forms ofthe antibody comprising a light chain amino acid residue deamidated atasparagine 31.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; and b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31. In the composition theheavy chain variable region may comprise an amino acid sequence havingat least 90%, 95% or 95.57% identity to the amino acid sequence of SEQID NO: 3. In the composition the light chain variable region maycomprise an amino acid sequence having at least 90%, 98% or 98.31%identity to the amino acid sequence of SEQ ID NO: 3.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; and b) oxidized forms of the antibody comprising at least oneselected from the group consisting of a heavy chain amino acid residueoxidized at tryptophan 52, a heavy chain amino acid residue oxidized atmethionine 64, a heavy chain amino acid residue oxidized at methionine82, a heavy chain amino acid residue oxidized at methionine 85 and alight chain amino acid residue oxidized at methionine 4.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain variable region sequencehaving the amino acid sequence shown in SEQ ID NO: 3 and a light chainvariable region sequence having the amino acid sequence shown in SEQ IDNO: 4; b) deamidated forms of the antibody comprising a light chainamino acid residue deamidated at asparagine 31; and c) oxidized forms ofthe antibody comprising at least one selected from the group consistingof a heavy chain amino acid residue oxidized at tryptophan 52, a heavychain amino acid residue oxidized at methionine 64, a heavy chain aminoacid residue oxidized at methionine 82, a heavy chain amino acid residueoxidized at methionine 85 and a light chain amino acid residue oxidizedat methionine 4.

In another embodiment the total protein concentration is about 75 mg/mL.The total protein concentration may also be about any pair of values, orsingle value in the range of about 75 mg/mL to about 150 mg/mL such asabout 75 mg/mL to about 100 mg/mL, about 67.3 to about 87.5 mg/mL, about76 g protein/L to about 82 g protein/L, about 46 g protein/L to about 66g protein/L or about 100 mg/mL. In the compositions the purity of theanti-human-IL-5 antibodies in the sample is greater than, or equal to,97.0%, 96%, 95%, or 80%, 85%.

In another embodiment the composition further comprises a) a main formof the antibody comprising greater than, or equal to, 50% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition.

In another embodiment the composition further comprises a) a main formof the antibody comprising greater than, or equal to, 50% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition; and b) acidic forms of the antibody comprising about20% to about 45% of the protein in the composition as measured usingcapillary isoelectric focusing of the composition.

In another embodiment the composition further comprises a) a main formof the antibody comprising greater than, or equal to, 50% of the proteinin the composition as measured using capillary isoelectric focusing ofthe composition; and b) a basic form of the antibody comprising about 1%to about 15% of the protein in the composition as measured usingcapillary isoelectric focusing of the composition.

In another embodiment the composition of further comprises a) a mainform of the antibody comprising greater than, or equal to, 50% of theprotein in the composition as measured using capillary isoelectricfocusing of the composition; b) acidic forms of the antibody comprisingabout 20% to about 45% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) a basicform of the antibody comprising about 1% to about 15% of the protein inthe composition as measured using capillary isoelectric focusing of thecomposition.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of an amino terminal pyroglutamate residue at aminoacid residue 1, a carboxy terminal glycine amino acid residue at aminoacid residue 448, a deamidated asparagine residue at position 299, adeamidated asparagine residue at position 317, a deamidated asparagineresidue at position 386, an oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine residue at position 222, an oxidized methionine atposition 254, an oxidized methionine at position 360 and an oxidizedmethionine residue at position 430; and b) a modified form of theantibody light chain amino acid sequence shown in SEQ ID NO: 2comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated asparagine residue at amino acidresidue 31, an oxidized methionine residue at position 4 and an oxidizedcysteine residue at position 220.

In another embodiment the composition comprises a) about greater than orequal to 92% of the population comprises an amino terminal pyroglutamateresidue at amino acid residue 1 of the antibody heavy chain, b) aboutgreater than or equal to 90% of the population comprises a carboxyterminal glycine amino acid residue at amino acid residue 448 of theantibody heavy chain, c) less than or equal to 6.0% of the populationcomprises a deamidated asparagine residue at position 386 of theantibody heavy chain; d) about less than or equal to 1.5% of thepopulation comprises an oxidized methionine residue at position 64 ofthe antibody heavy chain, e) about less than or equal to 4.5% of thepopulation comprises an oxidized methionine at position 254 of theantibody heavy chain, f) about less than or equal to 0.8% of thepopulation comprises an oxidized methionine residue at position 430 ofthe antibody heavy chain, and g) about less than or equal to 6.6% of thepopulation comprises a deamidated asparagine residue at amino acidresidue 31 of the antibody light chain.

In another embodiment the composition comprises a) about 92% to about99% of the population comprises an amino terminal pyroglutamate residueat amino acid residue 1 of the antibody heavy chain, b) about 95% toabout 99.5% of the population comprises a carboxy terminal glycine aminoacid residue at amino acid residue 448 of the antibody heavy chain, c)about 0.3% to about 1.5% of the population comprises a deamidatedasparagine residue at position 317 of the antibody heavy chain, d) about1.5% to about 4.5% of the population comprises a deamidated asparagineresidue at position 386 of the antibody heavy chain; e) about 0.5% toabout 1.5% of the population comprises an oxidized methionine residue atposition 64 of the antibody heavy chain, f) about 0.2% to about 1.5% ofthe population comprises an oxidized methionine residue at position 82of the antibody heavy chain or an oxidized methionine residue atposition 85 of the antibody heavy chain, g) about 2.5% to about 3.5% ofthe population comprises an oxidized methionine at position 254 of theantibody heavy chain, h) about 0.4% to about 0.8% of the populationcomprises an oxidized methionine residue at position 430 of the antibodyheavy chain, i) about 3.3% to about 6.6% of the population comprises adeamidated asparagine residue at amino acid residue 31 of the antibodylight chain, and j) about 0.1% to about 1% of the population comprisesan oxidized methionine residue at position 4 of the antibody lightchain.

In another embodiment the composition comprises: a) about 93.7% to about98.6% of the population comprises an amino terminal pyroglutamateresidue at amino acid residue 1 of the antibody heavy chain, b) about97.6% to about 99.2% of the population comprises a carboxy terminalglycine amino acid residue at amino acid residue 448 of the antibodyheavy chain, c) about 0.4% to about 1.2% of the population comprises adeamidated asparagine residue at position 317 of the antibody heavychain, d) about 1.6% to about 4.2% of the population comprises adeamidated asparagine residue at position 386 of the antibody heavychain; e) about 0.7% to about 0.9% of the population comprises anoxidized methionine residue at position 64 of the antibody heavy chain,f) about 0.3% to about 1.1% of the population comprises an oxidizedmethionine residue at position 82 of the antibody heavy chain or anoxidized methionine residue at position 85 of the antibody heavy chain,g) about 2.6% to about 3.3% of the population comprises an oxidizedmethionine at position 254 of the antibody heavy chain, h) about 0.5% toabout 0.7% of the population comprises an oxidized methionine residue atposition 430 of the antibody heavy chain, i) about 3.4% to about 6.5% ofthe population comprises a deamidated asparagine residue at amino acidresidue 31 of the antibody light chain, and j) about 0.2% to about 0.8%of the population comprises an oxidized methionine residue at position 4of the antibody light chain.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated asparagine residue at position 299,a deamidated asparagine residue at position 317, a deamidated asparagineresidue at position 386, an oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine residue at position 222, an oxidized methionine atposition 254, an oxidized methionine at position 360 and an oxidizedmethionine residue at position 430; and b) a modified form of theantibody light chain amino acid sequence shown in SEQ ID NO: 2comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated asparagine residue at amino acidresidue 31, an oxidized methionine residue at position 4 and an oxidizedcysteine residue at position 222.

In another embodiment the composition comprises: a) about 0.3% to about1.5% of the population comprises a deamidated asparagine residue atposition 317 of the antibody heavy chain, b) about 1.5% to about 4.5% ofthe population comprises a deamidated asparagine residue at position 386of the antibody heavy chain; c) about 0.5% to about 1.5% of thepopulation comprises an oxidized methionine residue at position 64 ofthe antibody heavy chain, d) about 0.2% to about 1.5% of the populationcomprises an oxidized methionine residue at position 82 of the antibodyheavy chain or an oxidized methionine residue at position 85 of theantibody heavy chain, e) about 2.5% to about 3.5% of the populationcomprises an oxidized methionine at position 254 of the antibody heavychain, f) about 0.4% to about 0.8% of the population comprises anoxidized methionine residue at position 430 of the antibody heavy chain,g) about 3.3% to about 6.6% of the population comprises a deamidatedasparagine residue at amino acid residue 31 of the antibody light chain,and h) about 0.1% to about 1% of the population comprises an oxidizedmethionine residue at position 4 of the antibody light chain.

In another embodiment the composition comprises: a) about 0.4% to about1.2% of the population comprises a deamidated asparagine residue atposition 317 of the antibody heavy chain, b) about 1.6% to about 4.2% ofthe population comprises a deamidated asparagine residue at position 386of the antibody heavy chain; c) about 0.7% to about 0.9% of thepopulation comprises an oxidized methionine residue at position 64 ofthe antibody heavy chain, d) about 0.3% to about 1.1% of the populationcomprises an oxidized methionine residue at position 82 of the antibodyheavy chain or an oxidized methionine residue at position 85 of theantibody heavy chain, e) about 2.6% to about 3.3% of the populationcomprises an oxidized methionine at position 254 of the antibody heavychain, f) about 0.5% to about 0.7% of the population comprises anoxidized methionine residue at position 430 of the antibody heavy chain,g) about 3.4% to about 6.5% of the population comprises a deamidatedasparagine residue at amino acid residue 31 of the antibody light chain,and h) about 0.2% to about 0.8% of the population comprises an oxidizedmethionine residue at position 4 of the antibody light chain.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of a deamidated at asparagine residue at position299, a deamidated asparagine residue at position 317 and a deamidatedasparagine residue at position 386; and b) a modified form of theantibody light chain amino acid sequence shown in SEQ ID NO: 2comprising a deamidated asparagine residue at amino acid residue 31.

In another embodiment the composition comprises: a) about 0.3% to about1.5% of the population comprises a deamidated asparagine residue atposition 317 of the antibody heavy chain, b) about 1.5% to about 4.5% ofthe population comprises a deamidated asparagine residue at position 386of the antibody heavy chain; and c) about 3.3% to about 6.6% of thepopulation comprises a deamidated asparagine residue at amino acidresidue 31 of the antibody light chain.

In another embodiment the composition comprises: a) about 0.4% to about1.2% of the population comprises a deamidated asparagine residue atposition 317 of the antibody heavy chain, b) about 1.6% to about 4.2% ofthe population comprises a deamidated asparagine residue at position 386of the antibody heavy chain; and c) about 3.4% to about 6.5% of thepopulation comprises a deamidated asparagine residue at amino acidresidue 31 of the antibody light chain.

Another aspect of the disclosure is a composition comprising apopulation of anti-IL-5 antibodies having a) a modified form of theantibody heavy chain amino acid sequence shown in SEQ ID NO: 1comprising at least one amino acid residue modification selected fromthe group consisting of an oxidized tryptophan residue at position 52,an oxidized methionine residue at position 64, an oxidized methionineresidue at position 82, an oxidized methionine residue at position 85,an oxidized cysteine residue at position 222, an oxidized methionine atposition 254, an oxidized methionine at position 360 and an oxidizedmethionine residue at position 430; and b) a modified form of theantibody light chain amino acid sequence shown in SEQ ID NO: 2comprising at least one amino acid residue modification selected fromthe group consisting of an oxidized methionine residue at position 4 andan oxidized cysteine residue at position 220.

In another embodiment the composition comprises: a) about 0.5% to about1.5% of the population comprises an oxidized methionine residue atposition 64 of the antibody heavy chain, b) about 0.2% to about 1.5% ofthe population comprises an oxidized methionine residue at position 82of the antibody heavy chain or an oxidized methionine residue atposition 85 of the antibody heavy chain, c) about 2.5% to about 3.5% ofthe population comprises an oxidized methionine at position 254 of theantibody heavy chain, d) about 0.4% to about 0.8% of the populationcomprises an oxidized methionine residue at position 430 of the antibodyheavy chain, and e) about 0.1% to about 1% of the population comprisesan oxidized methionine residue at position 4 of the antibody lightchain.

In another embodiment the composition comprises: a) about 0.7% to about0.9% of the population comprises an oxidized methionine residue atposition 64 of the antibody heavy chain, b) about 0.3% to about 1.1% ofthe population comprises an oxidized methionine residue at position 82of the antibody heavy chain or an oxidized methionine residue atposition 85 of the antibody heavy chain, c) about 2.6% to about 3.3% ofthe population comprises an oxidized methionine at position 254 of theantibody heavy chain, d) about 0.5% to about 0.7% of the populationcomprises an oxidized methionine residue at position 430 of the antibodyheavy chain, and e) about 0.2% to about 0.8% of the population comprisesan oxidized methionine residue at position 4 of the antibody lightchain.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; and b) a main form of the antibody comprisinggreater than, or equal to, 20% of the protein in the composition asmeasured using capillary isoelectric focusing of the composition.

Another aspect of the disclosure is a composition comprising a) ananti-IL-5 antibody comprising a heavy chain sequence having at least 90%identity to the amino acid sequence shown in SEQ ID NO: 1 and a lightchain sequence having at least 90% identity to the amino acid sequenceshown in SEQ ID NO: 2; b) a main form of the antibody comprising greaterthan, or equal to, 20% of the protein in the composition as measuredusing capillary isoelectric focusing of the composition; and c) acidicforms of the antibody comprising up to about 80% of the protein in thecomposition as measured using capillary isoelectric focusing of thecomposition.

In another embodiment the composition is for the treatment of a diseaseselected from the group consisting of asthma, severe eosinophilicasthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilicasthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease,eosinophilic granulomatosis with polyangiitis, hypereosinophilicsyndrome, nasal polyposis, bullous pemphigoid and eosinophilicesophagitis.

Another embodiment is a method of treating a disease in a subjectcomprising the steps of a) identifying a subject with a disease selectedfrom the group consisting of of asthma, severe eosinophilic asthma,severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma,sub-eosinophilic asthma, chronic obstructive pulmonary disease,eosinophilic granulomatosis with polyangiitis, hypereosinophilicsyndrome, nasal polyposis, bullous pemphigoid and eosinophilicesophagitis; and b) administering a therapeutically effective amount ofa composition according to the disclosure to the subject; whereby thedisease in the subject is treated.

Another embodiment is a method of producing a composition of thedisclosure, comprising the steps of: a) expressing in a host cell anantibody having a heavy chain amino acid sequence as shown in SEQ ID NO:1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or anantibody variant having a heavy chain amino acid sequence at least 90%identical to the heavy chain amino acid sequence and/or a light chainamino acid sequence at least 90% identical to the light chain amino acidsequence; b) growing the cells at a pH of about 6.75 to about 7.00; c)harvesting a cell culture supernatant; d) placing the cell culturesupernatant in contact with a protein A resin or protein G resin to bindantibody molecules; e) eluting the antibody molecules from the resin toproduce an first eluate; f) treating the first eluate at a pH of about3.3 to about 3.7 for about 15 to about 240 minutes to produce a treatedfirst eluate; g) placing the treated first eluate in contact with aanion exchange resin at a load pH of about 8.3 to about 8.7; h)collecting a second eluate (a flow through eluate) from the anionexchange resin and holding this for about 96 hours or less; i) treatingthe second eluate with guanidine and ammonium sulphate to produce asolution; j) placing the solution in contact with a hydrophobicinteraction chromatographic resin bed at a load ratio of about 12 gprotein/L resin to about 27 g protein/L of resin load ratio; k) elutinga third eluate comprising the antibody molecules from the hydrophobicinteraction chromatographic resin with an elution gradient volume ofabout 9 resin bed volumes to about 11 resin bed volumes and an elutionpeak cut stop of about 17% of the maximum peak height to about 23% ofthe maximum peak height; and 1) formulating the third eluate; whereby acomposition of the disclosure is produced. In the methods of thedisclosure any nucleic acid sequence suitable for expression of anantibody having a heavy chain amino acid sequence as shown in SEQ ID NO:1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or anantibody variant having a heavy chain amino acid sequence at least 90%identical to the heavy chain amino acid sequence and/or a light chainamino acid sequence at least 90% identical to the light chain amino acidsequence. For example, the nucleic acid sequence of SEQ ID NO: 13 or SEQID NO: 14 may be used to express an antibody in a eukaryotic cell.Alternatively, other nucleic acid sequences with different sequenceswhich encode (e.g., due to the use of alternative codons) the antibodyheavy chain amino acid sequence as shown in SEQ ID NO: 1 or the antibodylight chain amino acid sequence as shown in SEQ ID NO: 2 may be used. Inthe methods of the disclosure deamidation can be controlled by growingcells at a pH of about 6.75 to about 7.00. In the methods of thedisclosure deamidation can be controlled by growing cells for about 12to about 18 days for an in vitro cell age of less than or equal to 166days. In the methods of the disclosure deamidation can be controlled byplacing the treated first eluate in contact with an anion exchange resinat a load pH of about 8.3 to about 8.7 and collecting the second eluatefrom the anion exchange resin and holding this for about 96 hours orless. In the methods of the disclosure aggregationjcan be controlledduring phenyl SEPHAROSE™ fast flow chromatography by placing thesolution in contact with the hydrophobic interaction chromatographicresin bed at a load ratio of about 12 g protein/L resin to about 27 gprotein/L of resin load ratio; eluting a third eluate comprising theantibody molecules from the hydrophobic interaction chromatographicresin with an elution gradient volume of about 9 resin bed volumes toabout 11 resin bed volumes and an elution peak cut stop of about 17% ofthe maximum peak height to about 23% of the maximum peak height.Aggregation can also be limited after final filtration, filling andfreezing of the pharmaceutical compositions of the disclosure to lessthan or equal to about 6 hours. Importantly, any of the steps of thedisclosed methods may be omitted, or combined to produce thecompositions of the disclosure.

Upon production of the antibody, post-translational modifications mayoccur. This may include the cleavage of certain leader sequences, theaddition of various sugar moieties in various glycosylation patterns,deamidation (for example at an asparagine or glutamine residue),oxidation (for example at a methionine, tryptophan or free cysteineresidue), disulfide bond scrambling, isomerisation (for example at anaspartic acid residue), C-terminal lysine clipping (for example from oneor both heavy chains), and N-terminal glutamine cyclisation (for examplein the heavy and/or light chain).

The antibody composition may comprise (i) the antibody (i.e., anantibody having a heavy chain amino acid sequence as shown in SEQ ID NO:1 and a light chain amino acid sequence as shown in SEQ ID NO: 2); and(ii) antibody variants that include one or more or a combination of:charge variants (e.g., acidic and basic variants), amino acid sequencevariants, and antibody structural variants (e.g., aggregated andfragmented variants).

Acidic or basic antibody variants can be characterised and distinguishedfrom the antibody based on their overall acidic or basic charge. Forexample, the charge distribution of the antibody composition can bedetected using capillary isoelectric focussing (cIEF) or ion exchangechromatography. Acidic variants may comprise deamidated antibodyvariants, glycated antibody variants, sialylated antibody variants, andoxidised antibody variants. Cysteine and tryptophan oxidation in theantibody variant result in a pI shift (i.e., a charge difference) andare detected with other acidic antibody variants. Methionine oxidationin the antibody variant can be monitored by a change in antigen binding,or by peptide mapping, for example by LC-MS/MS.

Deamidation is an enzymatic reaction primarily converting asparagine (N)to iso-aspartic acid (iso-aspartate) (iso-D) and aspartic acid(aspartate) (D) at approximately 3:1 ratio. This deamidation reaction istherefore related to isomerization of aspartate (D) to iso-aspartate.The deamidation of asparagine and the isomerisation of aspartate, bothinvolve the intermediate succinimide. To a much lesser degree,deamidation can occur with glutamine residues in a similar manner.Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fcregion.

Deamidation causes a change in the charge of the antibody, such thatdeamidated antibody variants are acidic compared to the antibody. Theantibody composition may comprise ≤35% deamidated antibody variant. Forexample, N31 of the light chain may be deamidated to Iso-D, D orsuccinimide. The antibody composition may comprise ≤25% deamidatedantibody variant at position 31 of the light chain. This can result inone amino acid change in the sequence of the light chain of theantibody, for example in ≤25% of the antibody composition.

For example, N386 of the heavy chain may be deamidated to Iso-D, D orsuccinimide. The antibody composition may comprise ≤35% deamidatedantibody variant at position 386 of the heavy chain. This can result inone amino acid change in the sequence of the heavy chain of theantibody, for example in ≤35% of the antibody composition. Thecomposition may comprise a mixture of antibody variants. Deamidationevents can be cumulative, so that two or more asparagines residues aredeamidated. Therefore, the antibody composition may comprise at leastone amino acid change in the sequence of the heavy chain of the antibodyand/or at least one amino acid change in the sequence of the heavy chainof the antibody. For example, the antibody composition may comprisedeamidated antibody variant at position 31 of the light chain anddeamidated antibody variant at position 386 of the heavy chain.

Oxidation can occur during production and storage (i.e., in the presenceof oxidizing conditions) and results in a covalent modification of aprotein, induced either directly by reactive oxygen species orindirectly by reaction with secondary by-products of oxidative stress.Oxidation happens primarily with methionine residues, but may occur attryptophan and free cysteine residues. Oxidation can occur in a CDR, ina Fab (non-CDR) region, or in the Fc region.

Oxidation can cause a change in the charge of the antibody, such thatoxidised antibody variants are acidic compared to the antibody. Someoxidised antibody variants have the same charge as the antibody. Theantibody composition may comprise ≤55% oxidised antibody variant. Forexample, any one or a combination of M64, M254, and/or M430 of the heavychain may be oxidised. The antibody composition may comprise ≤55%oxidised antibody variant at any one or a combination of M64, M254,and/or M430 of the heavy chain. For example, W52 of the heavy chain maybe oxidised. The antibody composition may comprise ≤3% oxidised antibodyvariant at W52 of the heavy chain.

The composition may comprise a mixture of antibody variants. Therefore,the antibody composition may comprise at least one amino acid change inthe sequence of the heavy chain of the antibody and/or at least oneamino acid change in the sequence of the heavy chain of the antibody.For example, the antibody composition may comprise deamidated antibodyvariant at position 31 of the light chain; and/or deamidated antibodyvariant at position 386 of the heavy chain; and/or oxidation at any oneor a combination of M64, M254, and/or M430 and/or W52 of the heavychain.

Disulfide bond scrambling can occur during production and basic storageconditions. Under certain circumstances, disulfide bonds can break orform incorrectly, resulting in unpaired cysteine residues (—SH). Thesefree (unpaired) sulfhydryls (—SH) can promote shuffling.

N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in the heavychain and/or light chain is likely to form pyroglutamate (pGlu) viacyclization. It is thought that most pGlu formation happens in theproduction bioreactor, but it can also be formed non-enzymatically,depending on pH and temperature of processing and storage conditions.Cyclization of N-terminal Q or E is commonly observed in natural humanantibodies. The antibody composition described herein may comprise ≥50%pGlu at the N-terminus of the antibody. pGlu may be present in the heavychain. This can result in one amino acid change in the sequence of theheavy or light chain of the antibody, for example in ≥50% of theantibody composition.

The composition may comprise a mixture of antibody variants. Sequencechanges can be cumulative, so that the composition comprises two or moresequence changes in the heavy and/or light chain. Therefore, theantibody composition may comprise at least one amino acid change in thesequence of the heavy chain of the antibody and/or at least one aminoacid change in the sequence of the heavy chain of the antibody. Forexample, the antibody composition may comprise deamidated antibodyvariant at position 31 of the light chain; and/or deamidated antibodyvariant at position 386 of the heavy chain; and/or oxidation at any oneor a combination of M64, M254, and/or M430 and/or W52 of the heavychain; and/or pGlu at the N-terminus of the heavy and/or light chain.

C-terminal lysine clipping is an enzymatic reaction catalyzed bycarboxypeptidases, and is commonly observed in recombinant and naturalhuman antibodies. Variants of this process include removal of lysinefrom one or both heavy chains due to cellular enzymes from therecombinant host cell. Upon administration to the human subject/patientis likely to result in the removal of any remaining C-terminal lysines.The antibody composition described herein may comprise ≥50% C-terminallysine deleted at the C-terminus of the antibody. K449 may be deleted inone or both of the heavy chains of the antibody. Thus there are twoantibody variants: lysine single deletion in the heavy chain, and lysinedouble deletion in the heavy chain. The antibody (i.e., an antibodyhaving a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and alight chain amino acid sequence as shown in SEQ ID NO: 2) has bothlysines intact/present. This can result in one amino acid change in thesequence of the heavy chain of the antibody, for example in ≥50% of theantibody composition.

The composition may comprise a mixture of antibody variants. Forexample, the antibody composition may comprise deamidated antibodyvariant at position 31 of the light chain; and/or deamidated antibodyvariant at position 386 of the heavy chain; and/or oxidation at any oneor a combination of M64, M254, and/or M430 and/or W52 of the heavychain; and/or pGlu at the N-terminus of the heavy and/or light chain;and/or C-terminal lysine deleted at the C-terminus.

Aggregated or fragmented antibody variants can be characterised anddistinguished from the antibody based on their size. For example, thesize distribution of the antibody composition can be detected using sizeexclusion chromatography (SEC).

The antibody composition may comprise ≤20% aggregated antibody variant.The aggregated antibody variant may comprise dimer. The composition maycomprise a mixture of antibody variants. For example, the antibodycomposition may comprise deamidated antibody variant at position 31 ofthe light chain; and/or deamidated antibody variant at position 386 ofthe heavy chain; and/or oxidation at any one or a combination of M64,M254, and/or M430 and/or W52 of the heavy chain; and/or pGlu at theN-terminus of the heavy and/or light chain; and/or C-terminal lysinedeleted at the C-terminus; and/or aggregated antibody variant.

The compositions described may have been subjected to, or haveundergone, one or more post-translational modifications. Themodification may occur in a CDR, the variable framework region, or theconstant region. The modification may result in a change in charge ofthe molecule. The post-translational modifications and changes inprimary amino acid sequence described above, do not result insignificant changes in antigen binding affinity, biological activity,PK/PD, aggregation, immunogenicity, or binding to the Fc receptor, ofthe compositions. The compositions are substantially free ofcontaminating materials.

The antibody composition comprising the antibody and antibody variantsdescribed above retain specific antigen binding and/or FcRn binding. Forexample, the antibody composition comprising the antibody and antibodyvariants described above has ≥0.70 IL-5 specific antigen binding; and/or≥70% FcRn binding. Thus these levels (%) of variants can be tolerated inthe antibody composition without impacting function.

The compositions described herein may be produced by any number ofconventional techniques. For example, the compositions may be expressedin and purified from recombinant expression systems. In one embodiment,the composition is produced by a method of culturing a host cell underconditions suitable for expression of a polypeptide comprising SEQ IDNO: 1 and SEQ ID NO:2, wherein the composition is expressed, andoptionally purified, and optionally formulated within a pharmaceuticalcomposition.

A number of different expression systems and purification regimes can beused to produce the compositions. Generally, host cells are transformedwith a recombinant expression vector encoding the antibody. A wide rangeof host cells can be employed, including Eukaryotic cell lines ofmammalian origin (e.g., CHO, Perc6, HEK293, HeLa, NS0). Suitable hostcells include mammalian cells such as CHO (e.g., CHOK1 and CHO-DG44).

The host cell may be an isolated host cell. The host cell is usually notpart of a multicellular organism (e.g., plant or animal). The host cellmay be a non-human host cell.

Appropriate cloning and expression vectors for use with eukaryotic ormammalian cellular hosts and methods of cloning are known in the art.

The cells may be cultured under conditions that promote expression ofthe antibody. For example, a production bioreactor is used to culturethe cells. The production bioreactor volume may be: (i) about 20,000litres, about 10,000 litres; about 5,000 litres; about 2,000 litres;about 1,000 litres; or about 500 litres; or (ii) between 500 and 20,000litres; between 500 and 10,000 litres; between 500 and 5,000 litres;between 1,000 and 10,000 litres, or between 2,000 and 10,000 litres. Forexample, the cells may be cultured in a production bioreactor at a pH ofabout 6.75 to pH 7.00. Alternatively, the cells may be cultured in aproduction bioreactor for about 12 to about 18 days. Alternatively, thecells may be cultured in a production bioreactor at a pH of about 6.75to pH 7.00, for about 12 to about 18 days. This culture step may help tocontrol the level of deamidated antibody variants, for example, toreduce the level of deamidated antibody variants.

The composition may be recovered and purified by conventional proteinpurification procedures. For example, the composition may be harvesteddirectly from the culture medium. Harvest of the cell culture medium maybe via clarification, for example by centrifugation and/or depthfiltration. Recovery of the composition is followed by purification toensure adequate purity.

One or more chromatography steps may be used in purification, forexample one or more chromatography resins; and/or one or more filtrationsteps. For example affinity chromatography using resins, such as proteinA, G, or L may be used to purify the composition. Alternatively, or inaddition to, an ion-exchange resin such as a cation-exchange may be usedto purify the composition. Alternatively, or in addition to, ahydrophobic interaction chromatographic resin may be used to purify thecomposition. Alternatively the purification steps comprise: an affinitychromatography resin step, followed by a cation-exchange resin step,followed by a hydrophobic interaction chromatographic resin step.

For example, the harvest is placed in contact with a protein A resin.The solution comprising the composition may be eluted from the protein Aresin and treated at pH 3.3 to 3.7 for 15 to 240 minutes. This protein Aresin step may help to control the level of aggregated antibodyvariants, for example, to reduce the level of aggregated antibodyvariants.

The solution comprising the composition may then be further clarified bydepth filtration and/or dual layer filtration.

Alternatively, or in addition to, an anion exchange resin may be used.The solution comprising the composition may be placed in contact with ananion exchange resin (for example Q-SEPHAROSE™ Fast Flow anion exchangechromatography) at a load pH of 8.3 to 8.7. The solution comprising thecomposition may be eluted from the anion exchange resin and held for 96hours or less. This anion exchange resin step may help to control thelevel of deamidated antibody variants, for example, to reduce the levelof deamidated antibody variants.

Optionally, guanidine and/or ammonium sulphate may be added to thesolution comprising the composition, and held for 15 to 240 minutes.

Alternatively, or in addition to, a hydrophobic interactionchromatographic resin may be used. The solution comprising thecomposition may be placed in contact with a hydrophobic interactionchromatographic resin (e.g., phenyl SEPHAROSE™ fast flow chromatography)at a load ratio of 12 to 27 g protein/L resin. For example, the solutioncomprising the composition may be eluted using an elution gradientvolume (bed volumes; BV) of about 9 to about 11. An elution peak cutstop (% of maximum peak height) of about 17 to about 23 may be usedduring elution from the hydrophobic interaction chromatographic resin.This hydrophobic interaction chromatographic resin step may help tocontrol the level of aggregated antibody variants, for example, toreduce the level of aggregated antibody variants.

The solution comprising the composition may then be filtered to removevirus. The solution comprising the composition may then be formulated atan antibody concentration of about 76 g protein/L to about 82 gprotein/L, or to about 100 g protein/L. The solution comprising thecomposition may be filled into containers and frozen. Aliquots of thesolution comprising the composition may be lyophilized Lyophilizate maybe reconstituted by the addition of water to produce a compositioncomprising 75 mg/L of protein, the monoclonal anti-IL-5 mepolizumabantibody and 20 mM sodium phosphate dibasic heptahydrate, 12% weight ofsucrose to volume and 0.05% weight of polysorbate 80 to volume at a pHof from about 6.8 to about 7.2.

In another embodiment the composition of the disclosure is producedusing this method of producing a composition of the disclosure.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.2 to 6.6, wherein the buffering agent ishistidine, phosphate, citric acid, citric acid monohydrate, citrate or asalt thereof, wherein the purified preparation comprises the isoformsrepresented by peak 65, peak 78, peak 88, peak 92, the main peak andpeak 112 shown in FIG. 1, wherein the antibody comprises a heavy chainan amino acid sequence having at least 90% identity to the amino acidsequence of SEQ ID NO: 1 and a light chain amino acid sequence having atleast 90% identity to the amino acid sequence of SEQ ID NO: 2, andwherein the antibody is produced by a Chinese Hamster Ovary cell.

Another aspect of the disclosure is a composition comprising a purifiedpreparation of a monoclonal antibody and a buffering agent, wherein thecomposition is at a pH from 6.2 to 6.6, wherein the buffering agent isphosphate or a salt thereof, wherein the purified preparation comprisesthe isoforms represented by peak 65, peak 78, peak 88, peak 92, the mainpeak and peak 112 shown in FIG. 1, wherein the antibody comprises aheavy chain amino acid sequence having at least 90% identity to theamino acid sequence of SEQ ID NO: 1 and a light chain amino acidsequence having at least 90% identity to the amino acid sequence of SEQID NO: 2, and wherein the antibody is produced by a Chinese HamsterOvary cell.

In another embodiment of the compositions of the disclosure thebuffering agent is at least one selected from the group consisting ofsodium phosphate dibasic heptahydrate, phosphate and citric acid.

In another embodiment of the compositions of the disclosure comprise oneselected from a first formulation of 16.1 mM sodium phosphate dibasicheptahydrate, 3.9 mM citric acid, 12% weight of sucrose to volume, 0.02%weight of polysorbate 80 to volume and 0.05 mM EDTA; a secondformulation of 15.2 mM sodium phosphate dibasic heptahydrate, 4.8 mMcitric acid, 12% weight of sucrose to volume, 0.02% weight ofpolysorbate 80 to volume and 0.05 mM EDTA; a third formulation of 15.8mM sodium phosphate dibasic heptahydrate, 4.2 mM citric acid, 12% weightof sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05mM EDTA; a fourth formulation of 16.3 mM sodium phosphate dibasicheptahydrate, 3.7 mM citric acid, 12% weight of sucrose to volume, 0.02%weight of polysorbate 80 to volume and 0.05 mM EDTA; and a fifthformulation of 15.5 mM sodium phosphate dibasic heptahydrate, 4.5 mMcitric acid, 12% weight of sucrose to volume, 0.02% weight ofpolysorbate 80 to volume and 0.05 mM EDTA.

In summary, the disclosure includes:

1. A composition comprising an antibody having a heavy chain amino acidsequence as shown in SEQ ID NO: 1 and a light chain amino acid sequenceas shown in SEQ ID NO: 2, or an antibody variant having a heavy chainamino acid sequence at least 90% identical to the heavy chain amino acidsequence and/or a light chain amino acid sequence at least 90% identicalto the light chain amino acid sequence, wherein the compositioncomprises: ≤80% acidic antibody variants.2. The composition according to 1, wherein the composition has:

-   -   a) ≥0.70 IL-5 specific antigen binding; and/or    -   b) ≥70% FcRn binding.        3. The composition according to 2, wherein a) the specific        antigen binding is in the range of from 0.70 to 1.30; and/or b)        the FcRn binding is in the range of from 70% to 130%.        4. The composition according to any one of the preceding,        wherein the composition comprises: ≤35% deamidated antibody        variants.        5. The composition according to according to any one of the        preceding, wherein the composition comprises: ≤25% deamidated        antibody variants at N31 of the light chain amino acid sequence.        6. The composition according to according to any one of the        preceding, wherein the composition comprises: ≤35% deamidated        antibody variants at N386 of the heavy chain amino acid        sequence.        7. The composition according to any one of the preceding,        wherein the composition comprises: ≤55% oxidised antibody        variant at any one or a combination of:    -   a) M64 of the heavy chain amino acid sequence;    -   b) M254 of the heavy chain amino acid sequence; and/or    -   c) M430 of the heavy chain amino acid sequence.        8. The composition according to any one of the preceding,        wherein the composition comprises: ≤3% oxidised antibody        variants at W52 of the heavy chain amino acid sequence.        9. The composition according to any one of 4 to 8, wherein a        deamidated antibody variant amount and/or an oxidised variant        amount, is determined by peptide mapping LC-MS/MS.        10. The composition according to any one of the preceding,        wherein the composition comprises: ≤20% aggregated antibody        variants.        11. The composition according to 10, wherein the aggregated        antibody variant comprises a dimer.        12. The composition according to 10 or 11 wherein the aggregated        antibody variant amount is determined by SEC.        13. The composition according to any one of the preceding,        wherein the composition comprises: ≥50% heavy chain amino acid        sequence C-terminal lysine K449 deleted antibody variants.        14. The composition according to any one of the preceding,        wherein the composition comprises: ≥50% heavy chain amino acid        sequence pyro-glutamate N-terminal antibody variants.        15. A composition comprising an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence, wherein the composition comprises:        ≤80% acidic antibody variants and ≤20% aggregated antibody        variants.        16. A composition comprising an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence, wherein the composition comprises:        ≤25% deamidated antibody variants at N31 of the light chain        amino acid sequence; and ≤20% aggregated antibody variants.        17. A composition comprising an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence, wherein the composition comprises:        ≤25% deamidated antibody variants at N31 of the light chain        amino acid sequence; ≤55% oxidised antibody variants at M64 of        the heavy chain amino acid sequence; ≤3% oxidised antibody        variants at W52 of the heavy chain amino acid sequence; and ≤20%        aggregated antibody variants.        18. A composition comprising an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence, wherein the composition comprises:        ≤25% deamidated antibody variants at N31 of the light chain        amino acid sequence; ≤35% deamidated antibody variants at N386        of the heavy chain amino acid sequence; and ≤20% aggregated        antibody variants.        19. A composition comprising an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence, wherein the composition comprises:        ≤25% deamidated antibody variants at N31 of the light chain        amino acid sequence; ≤35% deamidated antibody variants at N386        of the heavy chain amino acid sequence; ≤55% oxidised antibody        variants at M64 of the heavy chain amino acid sequence, M254 of        the heavy chain amino acid sequence, M430 of the heavy chain        amino acid sequence; ≤3% oxidised antibody variants at W52 of        the heavy chain amino acid sequence; and ≤20% aggregated        antibody variants.        20. A composition comprising a purified preparation of a        monoclonal antibody and a buffering agent,        wherein the composition is at a pH from 6.8 to 7.2,        wherein the buffering agent is histidine, phosphate, citric        acid, citrate or a salt thereof,        wherein the purified preparation comprises the isoforms        represented by peak 65, peak 78, peak 88, peak 92, the main peak        and peak 112 shown in FIG. 1,        wherein the antibody comprises a heavy chain an amino acid        sequence having at least 90% identity to the amino acid sequence        of SEQ ID NO: 1 and a light chain amino acid sequence having at        least 90% identity to the amino acid sequence of SEQ ID NO: 2,        and        wherein the antibody is produced by a Chinese Hamster Ovary        cell.        21. The composition of 20, wherein the buffering agent is at        least one selected from the group consisting of sodium phosphate        dibasic heptahydrate, phosphate, citric acid and citrate.        22. The composition of 20, wherein the buffering agent is sodium        phosphate, potassium phosphate, or sodium citrate.        23. The composition of 20, wherein the composition further        comprises a sugar, a carbohydrate and/or a salt.        24. The composition of 23, wherein the composition comprises        sucrose.        25. A composition comprising a purified preparation of a        monoclonal antibody and a buffering agent,        wherein the composition is at a pH from 6.8 to 7.2,        wherein the buffering agent is phosphate or a salt thereof,        wherein the purified preparation comprises the isoforms        represented by peak 65, peak 78, peak 88, peak 92, the main peak        and peak 112 shown in FIG. 1,        wherein the antibody comprises a heavy chain amino acid sequence        having at least 90% identity to the amino acid sequence of SEQ        ID NO: 1 and a light chain amino acid sequence having at least        90% identity to the amino acid sequence of SEQ ID NO: 2, and        wherein the antibody is produced by a Chinese Hamster Ovary        cell.        26. The composition of 25, wherein the buffering agent is at        least one selected from the group consisting of sodium phosphate        dibasic heptahydrate, phosphate, citric acid and citrate.        27. The composition of 26, wherein the composition further        comprises a sugar.        28. The composition of 27, wherein the sugar is sucrose.        29. The composition of 28, comprising polysorbate 80.        30. The composition of 29, comprising one selected from a first        formulation of 20 mM sodium phosphate dibasic heptahydrate, 12%        weight of sucrose to volume and 0.05% weight of polysorbate 80        to volume; a second formulation of 15.5 mM sodium phosphate        dibasic heptahydrate, 3.9 mM citric acid monohydrate, 12% weight        of sucrose to volume, 0.02% weight of polysorbate 80 to volume        and 0.05 mM EDTA; and a third formulation of 26 mM sodium        phosphate dibasic heptahydrate, 15% weight of sucrose to volume        and 0.065% weight of polysorbate 80 to volume.        31. The composition of 29, wherein the antibody has a        dissociation constant equal to, or less than, about 3.5×10⁻¹¹ M        for human interleukin-5 comprising the amino acid sequence shown        in SEQ ID NO: 11.        32. The composition of 31, wherein the monoclonal antibody        concentration is about 75 mg/mL or about 100 mg/mL.        33. The composition of 30, wherein the antibody has a        dissociation constant equal to, or less than, about 3.5×10⁻¹¹ M        for human interleukin-5 comprising the amino acid sequence shown        in SEQ ID NO: 11.        34. The composition of 33, wherein the monoclonal antibody        concentration is about about 75 mg/mL or about 100 mg/mL.        35. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2; and    -   b) a main form of the antibody comprising greater than, or equal        to, 50% of the protein in the composition as measured using        capillary isoelectric focusing of the composition.        36. The composition of 35 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220.        37. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2;    -   b) a main form of the antibody comprising greater than, or equal        to, 50% of the protein in the composition as measured using        capillary isoelectric focusing of the composition; and    -   c) acidic forms of the antibody comprising about 20% to about        45% of the protein in the composition as measured using        capillary isoelectric focusing of the composition.        38. The composition of 37 wherein the acidic forms of the        antibody comprise at least one selected from the group        consisting of a peak 65 acidic form, a peak 78 acidic form, a        peak 88 acidic form and a peak 92 acidic form.        39. The composition of 38 wherein the acidic forms of the        antibody comprise at least one deamidated amino acid residue        selected from the group consisting of a heavy chain amino acid        residue deamidated at asparagine 299, a heavy chain amino acid        residue deamidated at asparagine 317, a heavy chain amino acid        residue deamidated at asparagine 386 and a light chain amino        acid residue deamidated at asparagine 31.        40. The composition of 39 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220.        41. The composition of 39 wherein the acidic forms of the        antibody comprise at least one oxidized amino acid residue        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a light chain amino acid        residue oxidized at cysteine 220.        42. The composition of 39 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220; and        wherein the acidic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        43. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2;    -   b) a main form of the antibody comprising greater than, or equal        to, 50% of the protein in the composition as measured using        capillary isoelectric focusing of the composition; and    -   c) a basic form of the antibody comprising about 1% to about 15%        of the protein in the composition as measured using capillary        isoelectric focusing of the composition.        44. The composition of 43 wherein the basic form of the antibody        comprises a peak 112 basic form.        45. The composition of 44 wherein the basic form of the antibody        comprises a heavy chain having a carboxy terminal residue that        is glycine 448.        46. The composition of 45 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220.        47. The composition of 45 wherein the basic forms of the        antibody comprise at least one oxidized amino acid residue        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a heavy chain amino acid        residue oxidized at cysteine 220.        48. The composition of 45 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a heavy chain amino acid residue        oxidized at cysteine 220; and        wherein the basic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        49. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2;    -   b) a main form of the antibody comprising greater than, or equal        to, 50% of the protein in the composition as measured using        capillary isoelectric focusing of the composition;    -   c) acidic forms of the antibody comprising about 20% to about        45% of the protein in the composition as measured using        capillary isoelectric focusing of the composition; and    -   d) a basic form of the antibody comprising about 1% to about 15%        of the protein in the composition as measured using capillary        isoelectric focusing of the composition.        50. The composition of 49 wherein the acidic forms of the        antibody comprise at least one selected from the group        consisting of a peak 65 acidic form, a peak 78 acidic form, a        peak 88 acidic form and a peak 92 acidic form.        51. The composition of 50 wherein the acidic forms of the        antibody comprise at least one deamidated amino acid residue        selected from the group consisting of a heavy chain amino acid        residue deamidated at asparagine 299, a heavy chain amino acid        residue deamidated at asparagine 317, a heavy chain amino acid        residue deamidated at asparagine 386 and a light chain amino        acid residue deamidated at asparagine 31.        52. The composition of 49 wherein the basic form of the antibody        comprises a peak 112 basic form.        53. The composition of 52 wherein the basic form of the antibody        comprises a heavy chain having a carboxy terminal residue that        is glycine 448.        54. The composition of 49 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220.        55. The composition of 49 wherein the acidic forms of the        antibody comprise at least one oxidized amino acid residue        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a heavy chain amino acid        residue oxidized at cysteine 220.        56. The composition of 49 wherein the basic forms of the        antibody comprise at least one oxidized amino acid residue        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a light chain amino acid        residue oxidized at cysteine 220.        57. The composition of 49 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a heavy chain amino acid residue        oxidized at cysteine 222; and        wherein the acidic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        58. The composition of 49 wherein the acidic forms of the        antibody comprise at least one oxidized amino acid residue        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a light chain amino acid        residue oxidized at cysteine 220; and        wherein the basic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        59. The composition of 49 wherein the main form of the antibody        comprise at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220; and        wherein the basic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        60. The composition of 49 wherein the main form of the antibody        comprises at least one oxidized amino acid residue selected from        the group consisting of a heavy chain amino acid residue        oxidized at tryptophan 52, a heavy chain amino acid residue        oxidized at methionine 64, a heavy chain amino acid residue        oxidized at methionine 82, a heavy chain amino acid residue        oxidized at methionine 85, a heavy chain amino acid residue        oxidized at cysteine 222, a heavy chain amino acid residue        oxidized at methionine 254, a heavy chain amino acid residue        oxidized at methionine 360, a heavy chain amino acid residue        oxidized at methionine 430, a light chain amino acid residue        oxidized at methionine 4 and a light chain amino acid residue        oxidized at cysteine 220;        wherein the acidic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 220, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220; and        wherein the basic forms of the antibody comprise at least one        oxidized amino acid residue selected from the group consisting        of a heavy chain amino acid residue oxidized at tryptophan 52, a        heavy chain amino acid residue oxidized at methionine 64, a        heavy chain amino acid residue oxidized at methionine 82, a        heavy chain amino acid residue oxidized at methionine 85, a        heavy chain amino acid residue oxidized at cysteine 222, a heavy        chain amino acid residue oxidized at methionine 254, a heavy        chain amino acid residue oxidized at methionine 360, a heavy        chain amino acid residue oxidized at methionine 430, a light        chain amino acid residue oxidized at methionine 4 and a light        chain amino acid residue oxidized at cysteine 220.        61. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2; and    -   b) deamidated forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue deamidated at asparagine 299, a heavy chain amino acid        residue deamidated at asparagine 317, a heavy chain amino acid        residue deamidated at asparagine 386 and a light chain amino        acid residue deamidated at asparagine 31.        62. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2; and    -   b) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a light chain amino acid        residue oxidized at cysteine 220.        63. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2;    -   b) deamidated forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue deamidated at asparagine 299, a heavy chain amino acid        residue deamidated at asparagine 317, a heavy chain amino acid        residue deamidated at asparagine 386 and a light chain amino        acid residue deamidated at asparagine 31; and    -   c) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85, a heavy chain amino acid        residue oxidized at cysteine 222, a heavy chain amino acid        residue oxidized at methionine 254, a heavy chain amino acid        residue oxidized at methionine 360, a heavy chain amino acid        residue oxidized at methionine 430, a light chain amino acid        residue oxidized at methionine 4 and a light chain amino acid        residue oxidized at cysteine 220.        64. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region having the CDRH1 amino acid sequence shown in SEQ ID NO:        5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the        CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light        chain variable region having the CDRL1 amino acid sequence shown        in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID        NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10;        and    -   b) deamidated forms of the antibody comprising a light chain        amino acid residue deamidated at asparagine 31.        65. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region having the CDRH1 amino acid sequence shown in SEQ ID NO:        5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the        CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light        chain variable region having the CDRL1 amino acid sequence shown        in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID        NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10;        and    -   b) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52 and a heavy chain amino acid        residue oxidized at methionine 64.        66. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region having the CDRH1 amino acid sequence shown in SEQ ID NO:        5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the        CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light        chain variable region having the CDRL1 amino acid sequence shown        in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID        NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10;    -   b) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52 and a heavy chain amino acid        residue oxidized at methionine 64; and    -   c) deamidated forms of the antibody comprising a light chain        amino acid residue deamidated at asparagine 31.        67. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region sequence having the amino acid sequence shown in SEQ ID        NO: 3 and a light chain variable region sequence having the        amino acid sequence shown in SEQ ID NO: 4; and    -   b) deamidated forms of the antibody comprising a light chain        amino acid residue deamidated at asparagine 31.        68. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region sequence having the amino acid sequence shown in SEQ ID        NO: 3 and a light chain variable region sequence having the        amino acid sequence shown in SEQ ID NO: 4; and    -   b) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85 and a light chain amino acid        residue oxidized at methionine 4.        69. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain variable        region sequence having the amino acid sequence shown in SEQ ID        NO: 3 and a light chain variable region sequence having the        amino acid sequence shown in SEQ ID NO: 4;    -   b) deamidated forms of the antibody comprising a light chain        amino acid residue deamidated at asparagine 31; and    -   c) oxidized forms of the antibody comprising at least one        selected from the group consisting of a heavy chain amino acid        residue oxidized at tryptophan 52, a heavy chain amino acid        residue oxidized at methionine 64, a heavy chain amino acid        residue oxidized at methionine 82, a heavy chain amino acid        residue oxidized at methionine 85 and a light chain amino acid        residue oxidized at methionine 4.        70. A composition comprising a population of anti-IL-5        antibodies having    -   a) a modified form of the antibody heavy chain amino acid        sequence shown in SEQ ID NO: 1 comprising at least one amino        acid residue modification selected from the group consisting of        an amino terminal pyroglutamate residue at amino acid residue 1,        a carboxy terminal glycine amino acid residue at amino acid        residue 448, a deamidated asparagine residue at position 299, a        deamidated asparagine residue at position 317, a deamidated        asparagine residue at position 386, an oxidized tryptophan        residue at position 52, an oxidized methionine residue at        position 64, an oxidized methionine residue at position 82, an        oxidized methionine residue at position 85, an oxidized cysteine        at position 222, an oxidized methionine at position 254, an        oxidized methionine at position 360 and an oxidized methionine        residue at position 430; and    -   b) a modified form of the antibody light chain amino acid        sequence shown in SEQ ID NO: 2 comprising at least one amino        acid residue modification selected from the group consisting of        a deamidated asparagine residue at amino acid residue 31, an        oxidized methionine residue at position 4 and an oxidized        cysteine at position 220.        71. The composition of 70 wherein:    -   a) about greater than or equal to 92% of the population        comprises an amino terminal pyroglutamate residue at amino acid        residue 1 of the antibody heavy chain,    -   b) about greater than or equal to 90% of the population        comprises a carboxy terminal glycine amino acid residue at amino        acid residue 448 of the antibody heavy chain,    -   c) less than or equal to 6.0% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain;    -   d) about less than or equal to 1.5% of the population comprises        an oxidized methionine residue at position 64 of the antibody        heavy chain,    -   e) about less than or equal to 4.5% of the population comprises        an oxidized methionine at position 254 of the antibody heavy        chain,    -   f) about less than or equal to 0.8% of the population comprises        an oxidized methionine residue at position 430 of the antibody        heavy chain, and    -   g) about less than or equal to 6.6% of the population comprises        a deamidated asparagine residue at amino acid residue 31 of the        antibody light chain.        72. The composition of 71 wherein:    -   a) about 93.7% to about 98.6% of the population comprises an        amino terminal pyroglutamate residue at amino acid residue 1 of        the antibody heavy chain,    -   b) about 97.6% to about 99.2% of the population comprises a        carboxy terminal glycine amino acid residue at amino acid        residue 448 of the antibody heavy chain,    -   c) about 0.4% to about 1.2% of the population comprises a        deamidated asparagine residue at position 317 of the antibody        heavy chain,    -   d) about 1.6% to about 4.2% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain;    -   e) about 0.7% to about 0.9% of the population comprises an        oxidized methionine residue at position 64 of the antibody heavy        chain,    -   f) about 0.3% to about 1.1% of the population comprises an        oxidized methionine residue at position 82 of the antibody heavy        chain or an oxidized methionine residue at position 85 of the        antibody heavy chain,    -   g) about 2.6% to about 3.3% of the population comprises an        oxidized methionine at position 254 of the antibody heavy chain,    -   h) about 0.5% to about 0.7% of the population comprises an        oxidized methionine residue at position 430 of the antibody        heavy chain,    -   i) about 3.4% to about 6.5% of the population comprises a        deamidated asparagine residue at amino acid residue 31 of the        antibody light chain, and    -   j) about 0.2% to about 0.8% of the population comprises an        oxidized methionine residue at position 4 of the antibody light        chain.        73. A composition comprising a population of anti-IL-5        antibodies having    -   a) a modified form of the antibody heavy chain amino acid        sequence shown in SEQ ID NO: 1 comprising at least one amino        acid residue modification selected from the group consisting of        a deamidated asparagine residue at position 299, a deamidated        asparagine residue at position 317, a deamidated asparagine        residue at position 386, an oxidized tryptophan residue at        position 52, an oxidized methionine residue at position 64, an        oxidized methionine residue at position 82, an oxidized        methionine residue at position 85, an oxidized cysteine at        position 222, an oxidized methionine at position 254, an        oxidized methionine at position 254, an oxidized methionine at        position 360 and an oxidized methionine residue at position 430;        and    -   b) a modified form of the antibody light chain amino acid        sequence shown in SEQ ID NO: 2 comprising at least one amino        acid residue modification selected from the group consisting of        a deamidated asparagine residue at amino acid residue 31, an        oxidized methionine residue at position 4 and an oxidized        cysteine at position 220.        74. The composition of 73 wherein:    -   a) about 0.3% to about 1.5% of the population comprises a        deamidated asparagine residue at position 317 of the antibody        heavy chain,    -   b) about 1.5% to about 4.5% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain;    -   c) about 0.5% to about 1.5% of the population comprises an        oxidized methionine residue at position 64 of the antibody heavy        chain,    -   d) about 0.2% to about 1.5% of the population comprises an        oxidized methionine residue at position 82 of the antibody heavy        chain or an oxidized methionine residue at position 85 of the        antibody heavy chain,    -   e) about 2.5% to about 3.5% of the population comprises an        oxidized methionine at position 254 of the antibody heavy chain,    -   f) about 0.4% to about 0.8% of the population comprises an        oxidized methionine residue at position 430 of the antibody        heavy chain,    -   g) about 3.3% to about 6.6% of the population comprises a        deamidated asparagine residue at amino acid residue 31 of the        antibody light chain, and    -   h) about 0.1% to about 1% of the population comprises an        oxidized methionine residue at position 4 of the antibody light        chain.        75. The composition of 74 wherein:    -   a) about 0.4% to about 1.2% of the population comprises a        deamidated asparagine residue at position 317 of the antibody        heavy chain,    -   b) about 1.6% to about 4.2% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain;    -   c) about 0.7% to about 0.9% of the population comprises an        oxidized methionine residue at position 64 of the antibody heavy        chain,    -   d) about 0.3% to about 1.1% of the population comprises an        oxidized methionine residue at position 82 of the antibody heavy        chain or an oxidized methionine residue at position 85 of the        antibody heavy chain,    -   e) about 2.6% to about 3.3% of the population comprises an        oxidized methionine at position 254 of the antibody heavy chain,    -   f) about 0.5% to about 0.7% of the population comprises an        oxidized methionine residue at position 430 of the antibody        heavy chain,    -   g) about 3.4% to about 6.5% of the population comprises a        deamidated asparagine residue at amino acid residue 31 of the        antibody light chain, and    -   h) about 0.2% to about 0.8% of the population comprises an        oxidized methionine residue at position 4 of the antibody light        chain.        76. A composition comprising a population of anti-IL-5        antibodies having    -   a) a modified form of the antibody heavy chain amino acid        sequence shown in SEQ ID NO: 1 comprising at least one amino        acid residue modification selected from the group consisting of        a deamidated asparagine residue at position 299, a deamidated        asparagine residue at position 317 and a deamidated asparagine        residue at position 386; and    -   b) a modified form of the antibody light chain amino acid        sequence shown in SEQ ID NO: 2 comprising a deamidated        asparagine residue at amino acid residue 31.        77. The composition of 76 wherein:    -   a) about 0.3% to about 1.5% of the population comprises a        deamidated asparagine residue at position 317 of the antibody        heavy chain,    -   b) about 1.5% to about 4.5% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain; and    -   c) about 3.3% to about 6.6% of the population comprises a        deamidated asparagine residue at amino acid residue 31 of the        antibody light chain.        78. The composition of 77 wherein:    -   a) about 0.4% to about 1.2% of the population comprises a        deamidated asparagine residue at position 317 of the antibody        heavy chain,    -   b) about 1.6% to about 4.2% of the population comprises a        deamidated asparagine residue at position 386 of the antibody        heavy chain; and    -   c) about 3.4% to about 6.5% of the population comprises a        deamidated asparagine residue at amino acid residue 31 of the        antibody light chain.        79. A composition comprising a population of anti-IL-5        antibodies having    -   a) a modified form of the antibody heavy chain amino acid        sequence shown in SEQ ID NO: 1 comprising at least one amino        acid residue modification selected from the group consisting of        an oxidized tryptophan residue at position 52, an oxidized        methionine residue at position 64, an oxidized methionine        residue at position 82, an oxidized methionine residue at        position 85, an oxidized cysteine at position 222, an oxidized        methionine at position 254, an oxidized methionine at position        360 and an oxidized methionine residue at position 430; and    -   b) a modified form of the antibody light chain amino acid        sequence shown in SEQ ID NO: 2 comprising at least one amino        acid residue modification selected from the group consisting of        an oxidized methionine residue at position 4 and an oxidized        cysteine at position 220.        80. The composition of 79 wherein:    -   c) about 0.5% to about 1.5% of the population comprises an        oxidized methionine residue at position 64 of the antibody heavy        chain,    -   d) about 0.2% to about 1.5% of the population comprises an        oxidized methionine residue at position 82 of the antibody heavy        chain or an oxidized methionine residue at position 85 of the        antibody heavy chain,    -   e) about 2.5% to about 3.5% of the population comprises an        oxidized methionine at position 254 of the antibody heavy chain,    -   f) about 0.4% to about 0.8% of the population comprises an        oxidized methionine residue at position 430 of the antibody        heavy chain, and    -   g) about 0.1% to about 1% of the population comprises an        oxidized methionine residue at position 4 of the antibody light        chain.        81. The composition of 80 wherein:    -   a) about 0.7% to about 0.9% of the population comprises an        oxidized methionine residue at position 64 of the antibody heavy        chain,    -   b) about 0.3% to about 1.1% of the population comprises an        oxidized methionine residue at position 82 of the antibody heavy        chain or an oxidized methionine residue at position 85 of the        antibody heavy chain,    -   c) about 2.6% to about 3.3% of the population comprises an        oxidized methionine at position 254 of the antibody heavy chain,    -   d) about 0.5% to about 0.7% of the population comprises an        oxidized methionine residue at position 430 of the antibody        heavy chain, and    -   e) about 0.2% to about 0.8% of the population comprises an        oxidized methionine residue at position 4 of the antibody light        chain.        82. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2; and    -   b) a main form of the antibody comprising greater than, or equal        to, 20% of the protein in the composition as measured using        capillary isoelectric focusing of the composition.        83. A composition comprising    -   a) an anti-IL-5 antibody comprising a heavy chain sequence        having at least 90% identity to the amino acid sequence shown in        SEQ ID NO: 1 and a light chain sequence having at least 90%        identity to the amino acid sequence shown in SEQ ID NO: 2;    -   b) a main form of the antibody comprising greater than, or equal        to, 20% of the protein in the composition as measured using        capillary isoelectric focusing of the composition; and    -   c) acidic forms of the antibody comprising up to about 80% of        the protein in the composition as measured using capillary        isoelectric focusing of the composition.        84. A composition according to any of the preceding for the        treatment of a disease selected from the group consisting of        asthma, severe eosinophilic asthma, severe asthma, uncontrolled        eosinophilic asthma, eosinophilic asthma, sub-eosinophilic        asthma, chronic obstructive pulmonary disease, eosinophilic        granulomatosis with polyangiitis, hypereosinophilic syndrome,        nasal polyposis, bullous pemphigoid and eosinophilic        esophagitis.        85. A method of treating a disease in a subject comprising the        steps of    -   a) identifying a subject with a disease selected from the group        consisting of of asthma, severe eosinophilic asthma, severe        asthma, uncontrolled eosinophilic asthma, eosinophilic asthma,        sub-eosinophilic asthma, chronic obstructive pulmonary disease,        eosinophilic granulomatosis with polyangiitis, hypereosinophilic        syndrome, nasal polyposis, bullous pemphigoid and eosinophilic        esophagitis; and    -   b) administering a therapeutically effective amount of a        composition according to any of the preceding to the subject;        whereby the disease in the subject is treated.        86. The composition of 20 to 83, wherein the composition has:    -   a) ≥0.70 IL-5 specific antigen binding; and/or    -   b) ≥70% FcRn binding.        87. The composition of 20 to 83, wherein a) the specific antigen        binding is in the range of from 0.70 to 1.30; and/or b) the FcRn        binding is in the range of from 70% to 130%.        88. The composition of 20 to 83, wherein the composition        comprises: ≤20% aggregated antibody variants.        89. The composition of 20 to 83, wherein the aggregated antibody        variant comprises a dimer.        90. A method of producing a composition of 1-83, comprising the        steps of:        a) expressing in a host cell an antibody having a heavy chain        amino acid sequence as shown in SEQ ID NO: 1 and a light chain        amino acid sequence as shown in SEQ ID NO: 2, or an antibody        variant having a heavy chain amino acid sequence at least 90%        identical to the heavy chain amino acid sequence and/or a light        chain amino acid sequence at least 90% identical to the light        chain amino acid sequence;        b) growing the cells at a pH of about 6.75 to about 7.00 for        about 12 to about 18 days for an in vitro cell age of less than        or equal to 166 days;        c) harvesting a cell culture supernatant;        d) placing the cell culture supernatant in contact with a        protein A resin or protein G resin to bind antibody molecules;        e) eluting the antibody molecules from the resin to produce an        first eluate;        f) treating the first eluate at a pH of about 3.3 to about 3.7        for about 15 to about 240 minutes to produce a treated first        eluate;        g) placing the treated first eluate in contact with a anion        exchange resin at a load pH of about 8.3 to about 8.7;        h) collecting a second eluate from the anion exchange resin and        holding this for about 96 hours or less;        i) treating the second eluate with guanidine and ammonium        sulphate to produce a solution;        j) placing the solution in contact with a hydrophobic        interaction chromatographic resin bed at a load ratio of about        12 g protein/L resin to about 27 g protein/L of resin load        ratio;        k) eluting a third eluate comprising the antibody molecules from        the hydrophobic interaction chromatographic resin with an        elution gradient volume of about 9 resin bed volumes to about 11        resin bed volumes and an elution peak cut stop of about 17% of        the maximum peak height to about 23% of the maximum peak height;        and        l) formulating the third eluate;        whereby the composition of 1-83 is produced.        91. The composition of 1-83 produced by the method of 90.        92. A composition comprising a purified preparation of a        monoclonal antibody and a buffering agent,        wherein the composition is at a pH from 6.2 to 6.6,        wherein the buffering agent is histidine, phosphate, citric        acid, citric acid monohydrate, citrate or a salt thereof,        wherein the purified preparation comprises the isoforms        represented by peak 65, peak 78, peak 88, peak 92, the main peak        and peak 112 shown in FIG. 1,        wherein the antibody comprises a heavy chain an amino acid        sequence having at least 90% identity to the amino acid sequence        of SEQ ID NO: 1 and a light chain amino acid sequence having at        least 90% identity to the amino acid sequence of SEQ ID NO: 2,        and        wherein the antibody is produced by a Chinese Hamster Ovary        cell.        93. The composition of 92, wherein the buffering agent is at        least one selected from the group consisting of sodium phosphate        dibasic heptahydrate, phosphate, citric acid and citric acid        monohydrate.        94. The composition of 92, wherein the buffering agent is sodium        phosphate, potassium phosphate, citric acid, citric acid        monohydrate or sodium citrate.        95. The composition of 92, wherein the composition further        comprises a sugar, a carbohydrate and/or a salt.        96. The composition of 95, wherein the composition comprises        sucrose.        97. A composition comprising a purified preparation of a        monoclonal antibody and a buffering agent,        wherein the composition is at a pH from 6.2 to 6.6,        wherein the buffering agent is phosphate or a salt thereof,        wherein the purified preparation comprises the isoforms        represented by peak 65, peak 78, peak 88, peak 92, the main peak        and peak 112 shown in FIG. 1,        wherein the antibody comprises a heavy chain amino acid sequence        having at least 90% identity to the amino acid sequence of SEQ        ID NO: 1 and a light chain amino acid sequence having at least        90% identity to the amino acid sequence of SEQ ID NO: 2, and        wherein the antibody is produced by a Chinese Hamster Ovary        cell.        98. The composition of 97, wherein the buffering agent is at        least one selected from the group consisting of sodium phosphate        dibasic heptahydrate, phosphate, citric acid, citric acid        monohydrate and citrate.        99. The composition of 98, wherein the composition further        comprises a sugar.        100. The composition of 99, wherein the sugar is sucrose.        101. The composition of 100, comprising polysorbate 80.        102. The composition of 101, comprising one selected from a        first formulation of 16.1 mM sodium phosphate dibasic        heptahydrate, 3.9 mM citric acid monohydrate, 12% weight of        sucrose to volume, 0.02% weight of polysorbate 80 to volume and        0.05 mM EDTA; a second formulation of 15.2 mM sodium phosphate        dibasic heptahydrate, 4.8 mM citric acid monohydrate, 12% weight        of sucrose to volume, 0.02% weight of polysorbate 80 to volume        and 0.05 mM EDTA; a third formulation of 15.8 mM sodium        phosphate dibasic heptahydrate, 4.2 mM citric acid monohydrate,        12% weight of sucrose to volume, 0.02% weight of polysorbate 80        to volume and 0.05 mM EDTA; a fourth formulation of 16.3 mM        sodium phosphate dibasic heptahydrate, 3.7 mM citric acid        monohydrate, 12% weight of sucrose to volume, 0.02% weight of        polysorbate 80 to volume and 0.05 mM EDTA; and a fifth        formulation of 15.5 mM sodium phosphate dibasic heptahydrate,        4.5 mM citric acid monohydrate, 12% weight of sucrose to volume,        0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA.        103. The composition of 101, wherein the antibody has a        dissociation constant equal to, or less than, about 3.5×10⁻¹¹ M        for human interleukin-5 comprising the amino acid sequence shown        in SEQ ID NO: 11.        104. The composition of 101, wherein the monoclonal antibody        concentration is about 75 mg/mL or about 100 mg/mL.        105. The composition of 102, wherein the antibody has a        dissociation constant equal to, or less than, about 3.5×10⁻¹¹ M        for human interleukin-5 comprising the amino acid sequence shown        in SEQ ID NO: 11.        106. A composition according to any one of the preceding wherein        the antibody is at a concentration of between about 75 mg/ml to        about 100 mg/ml.        107. A composition according to any one of the preceding wherein        the composition further comprises one or a combination of:    -   a) a buffering agent selected from the group consisting of        sodium phosphate dibasic heptahydrate, phosphate, citrate,        sodium phosphate, potassium phosphate, sodium citrate, and        histidine, providing a pH of between 6.8 and 7.2; and/or    -   b) a sugar; and/or    -   c) polysorbate 80; and/or d) EDTA.        108. A composition according to any one of the preceding claims        wherein the composition further comprises one or a combination        of:    -   a) a buffering agent selected from the group consisting of        sodium phosphate dibasic heptahydrate, phosphate, citrate,        citric acid monohydrate, sodium phosphate, potassium phosphate,        sodium citrate, and histidine, providing a pH of between 6.2 and        6.6; and/or    -   b) a sugar; and/or    -   c) polysorbate 80; and/or d) EDTA.

EXAMPLES Example 1 Preparation of a Composition

Multiple batches of a composition comprising the monoclonal anti-IL-5antibody mepolizumab were produced.

An inoculum of Chinese Hamster Ovary cells stably transfected withexpression vector constructs comprising the nucleic acid sequences shownSEQ ID NO: 13 and SEQ ID NO: 14 was cultured in 5000 L bioreactorscontaining a liquid cell culture medium. The mature antibody encoded bythese nucleic acids is mepolizumab and comprises the heavy chain aminoacid sequence shown in SEQ ID NO: 1 and the light chain amino acidsequence shown in SEQ ID NO: 2.

Bioreactors were operated at a temperature of about 34.5° C. to about35.5° C. Air and oxygen were sparged into the culture medium and a pH ofabout 6.75 to 7.00 was maintained. The culture duration was about 12 to18 days. The in vitro cell age (culture days from initial thaw of mastercell bank to harvest) was 166 days or less. After this, a clarified cellculture supernatant was harvested by centrifugation and depth filtrationof the cell culture medium. This clarified supernatant was thensubjected to protein A chromatography and impurities were allowed toflow off this chromatography column. Bound protein including antibodymolecules was then eluted from the protein A column, treated at a pH ofabout 3.3 to 3.7 for about 15 to 240 minutes. This treated preparationwas then adjusted to about pH 4.3 to 4.7 and held for about 20 to 1110minutes. This treated preparation was then clarified through thefiltration train of a depth filter and a 0.5/0.2 μm dual layer filter.The filtered preparation was then subjected to Q-SEPHAROSE™ Fast Flowanion exchange chromatography at a load pH of about 8.3 to 8.7 andeluted from the chromatography column. This eluate was then held forabout 96 hours or less. Guanidine and ammonium sulfate were then added.Guanidine was added to a concentration of about 1.8 M to 2.2 M and heldfor about 15 to 240 minutes. This solution was then subjected to phenylSEPHAROSE™ fast flow chromatography at a load ratio of about 12 gprotein/L resin to about 27 g/L resin, an elution gradient volume (bedvolumes; BV) of about 9 to about 11, and elution peak cut stop (% ofmaximum peak height) of about 17 to about 23. Virus filtration was thenperformed using a Planova 20N virus removal filter. This filtrate wasthen adjusted to a target concentration of about 46 g protein/L to about66 g protein/L and the bulk drug substances (BDS) were formulated bytangential filtration and ultrafiltration exchange with a solutioncomprising about 20 mM sodium phosphate dibasic heptahydrate and 12%weight of sucrose to volume. This solution was then adjusted to a targetconcentration of 76 g protein/L to about 82 g protein/L and about 0.05%weight of polysorbate 80 to volume was added. This solution was thenfiltered through 0.5/0.2 μm PES filters and containers of the solutionwere filled and frozen. Drug product was manufactured using a sterilemanufacturing process involving thawing and combining bulk containersfollowed by filtration of bulk into vials, lyophilization, stopperingand crimping by manufacturing processes well known in the art. The finaldrug product presentation is a lyophilized drug product in a single usevial.

Lyophilizate from each batch produced was reconstituted by the additionof water to produce a composition comprising 100 mg/mL of protein, themonoclonal anti-IL-5 mepolizumab antibody and 26 mM sodium phosphatedibasic heptahydrate, 15% weight of sucrose to volume and 0.065% weightof polysorbate 80 to volume at a pH of from about 6.8 to about 7.2.

Example 2 Characterization of the Composition

Samples from the batches of composition comprising a monoclonalanti-IL-5 antibody produced as described above were characterized.

Capillary isoelectric focusing (cIEF) consistently showed the presenceof six antibody isoforms in the composition (e.g., composition referencestandard (RS) 101245722). See FIG. 1. These isoforms are the peak 65,peak 78, peak 88, peak 92, main peak and peak 112 isoforms shown inFIG. 1. Samples of the composition were subjected to cIEF using standardmethods. pI 7.9 and pI 9.46 standards were included in samples to beanalyzed by cIEF. The cIEF electropherogram shown in FIG. 1 isrepresentative of those for the composition from multiple batches.

The electropherogram shows the composition comprises a main form, acidicforms and basic forms of the antibody. The main form can be seen in FIG.1 and is also identified as peak 100 in some instances. The acidic formsof the antibody correspond to the peak 65, peak 78, peak 88 and peak 92forms of FIG. 1. The basic forms of the antibody correspond to the peak112 forms of FIG. 1.

The formula used for assignment of peaks is as follows:

peakname=int{(pIPeak-8)/(pIMain-8)*100} where: int=integer and pIPeak=pIof the peak to be named. Table 7 shows the peak naming convention forcIEF electropherograms of the composition comprising a monoclonalanti-IL-5 antibody. The name of the peaks determined as described hereshould be verified based on the observed electrophoretic/chromatographicpattern. Peaks that do not fall within the ranges described here shouldbe processed according to the formula above.

TABLE 7 Identification of cIEF peaks Peak Peak Peak Peak Peak Peak PeakPeak Peaks with Retention 62 75 85 89 100 103 109 120 times greater thanRention 68 81 91 95 100 109 115 126 times less than Report Peak PeakPeak Peak Peak Peak Peak Peak as Peak 65 78 88 92 100 106 112 123

Integral analysis of the electropherogram peaks was performed. See Table8.

TABLE 8 Total peak area in Select cIEF Electropherograms of DifferentComposition Batches. MDS2 Batch MDS1 Batch T0414001 T0414002 T0414003T04L009 T04M001 T04N002 (PPQ¹1) (PPQ¹2) (PPQ¹3) Charge Isoforms by cIEFPeak area Peak area Peak area Peak area Peak area Peak area 61.2% for63.9% for 60.6% for 58.1% for 61.8% for 62.3% for main; main; main;main; main; main; 37.5% for 34.4% for 38.0% for 37.1% for 34.1% for33.0% for total total total total total total acidic; acidic; acidic;acidic; acidic; acidic; 1.2% for 1.6% for 1.3% for 4.9% for 4.0% for4.7% for total basic total basic total basic total basic total basictotal basicThis showed the main form represented greater than or equal to 50.0% ofthe total peak area in the samples (with values between from about 58.1%and 62.3% being observed as well). This also showed the acidic formsrepresented less than or equal to 45.0% of the total peak area in thesamples (with values of between from about 20% to about 45% such as32.2% and 40.7% being observed as well). The basic forms representedfrom about 1% to about 15% of the total peak area in the samples (withvalues of between from about 1.2% to about 4.9% being observed as well).

The main form, acidic forms and basic forms peak fractions produced bycIEF were then further analyzed by weak cation exchange (WCX), trypsinpeptide mapping and Liquid Chromatography-Mass Spectroscopy/MassSpectroscopy (LC-MS/MS) analyses. Standard methods were used for theseanalyses.

These WCX, trypsin peptide mapping and LC-MS/MS results showed that themain form peak fraction contained two IgG₁ mAb modifications. Thus, inthe antibody heavy chain amino acid sequence of SEQ ID NO: 99.6% of theN-terminal glutamine (Gln, Q) was cyclized to pyroglutamic acid (pGlu)and 99.9% of the heavy chain (HC) C-terminal lysine (Lys, K) 449 wascleaved. Typically, the pGlu levels in the batches tested were >95.0%and the level of HC without C-terminal K449 levels was ≥98.0%.

These WCX, trypsin peptide mapping and LC-MS/MS results also showed forthe acid forms peaks that a one-Dalton mass shift characteristic ofdeamidation was observed. Peptide mapping LC-MS/MS demonstrated theacidic forms peaks contain a mixture of deamidated antibody species.Deamidation was predominantly observed at HC N386 of the amino acidsequence shown in SEQ ID NO: 1 and at LC N31 of the amino acid sequenceshown in SEQ ID NO: 2. Lower levels of deamidation were also observed atHC N317 of the amino acid sequence shown in SEQ ID NO: 1.

In its entirety, this experimental data showed asparagine residues HCN317, HC N386, HC N299 of the amino acid sequence shown in SEQ ID NO: 1and LC N31 of the amino acid sequence shown in SEQ ID NO: 2 weresusceptible to deamidation.

These WCX, trypsin peptide mapping and LC-MS/MS showed for the basicforms peaks that the antibody forms in this peak had at least one heavychain, carboxy terminal lysine amino acid residue intact. Antibodyspecies with intact lysines, relative to other forms in which these areabsent, will migrate in the basic region due to additional positivecharges from these residues. Thus, the basic forms, such as peak 112,correspond to antibody forms in which one, or both, heavy chain aminoacid sequences have the carboxy terminal lysine amino acid sequenceshown in SEQ ID NO: 1 intact.

Primary sequencing of the composition comprising a monoclonal anti-IL-5antibody was also performed by standard LC-MS/MS techniques. Theseanalyses examined the primary struction and amino acid sequence of theantibody molecules in the composition. In particular, these analysesshowed which amino acid residues were deamidated, oxidized, cyclized orabsent in the anti-IL-5 antibody and the percentage of these in thepopulation of anti-IL-5 antibodies (e.g., expressed from the nucleicacid sequence of SEQ ID NO: 13 and the nucleic acid sequence of SEQ IDNO: 14) present in the composition. See Table 9.

TABLE 9 Primary antibody sequence by peptide mapping LC-MS/MS. MDS2Batch MDS1 Batch T0414001 T0414002 T0414003 T04L009 T04M001 T04N002(PPQ¹1) (PPQ¹2) (PPQ¹3) Primary Sequence by Peptide Mapping LC-MS/MSDeamidation Deamidation Deamidation Deamidation Deamidation Deamidation1.0% of heavy 1.1% of HC 1.1% of HC 1.1% of HC 1.2% of HC 1.1% of HCchain (HC or N317; N317; N317; N317; N317; H) asparagine (N) 317 ; 1.9%of HC 2.2% of HC 1.6% of HC 1.7% of HC 1.6% of HC 1.9% of HC N386; N386;N386; N386; N386; N386; 5.8% of light 6.5% of LC 6.2% of LC 5.6% of LC6.5% of LC 6.2% of LC chain (LC or L) N31 N31 N31 N31 N31 N31 HC 1-5pGlu HC 1-5 pGlu HC 1-5 pGlu HC1-5 pGlu HC 1-5 pGlu HC 1-5 93.7%; 94.6%;94.0%; 93.7%; 94.6%; pGlu 95.3%; HC 449 Lys HC 449 Lys HC 449 Lys HC 449Lys HC 449 Lys HC 449 Lys Clipped Clipped Clipped Clipped ClippedClipped 99.2% 98.4% 97.6% 99.2% 98.9% 98.5% Oxidation Oxidation =Oxidation = Oxidation = Oxidation = Oxidation = 0.9% of HC 1.0% of HC0.8% of HC 0.7% of HC 0.8% of HC 0.7% of HC methionine (M) M64; M64;M64; M64; M64; 64; 1.1% of HC 0.7% of HC 0.8% of HC 0.7% of HC 0.7% ofHC 0.7% of HC M82/85; M82/85 ; M82/85; M82/85; M82/85; M82/85; 3.0% ofHC 2.9% of HC 3.1% of HC 2.6% of HC 2.7% of HC 2.7% of HC M254; M254;M254; M254; M254; M254; 0.7% of HC 0.5% of HC 0.5% of HC 0.4% of HC 0.4%of HC 0.5% of HC M360; M360; M360; M360; M360; M360; 0.6% of HC 0.6% ofHC 0.6% of HC 0.5% of HC 0.5% of HC 0.6% of HC M430; M430; M430; M430;M430; M430; 0.8% of LC 0.4% of LC 0.5% of LC 0.3% of LC 0.4% of LC 0.5%of LC M4 M4 M4 M4 M4 M4

Antibody Variants

Mepolizumab binds to soluble IL-5 and blocks the soluble IL-5 frombinding to its receptor. The structure of IL-5 is indicative of asecreted protein and there is no evidence of any membrane-bound forms ofIL-5 on any cell types. Thus, the Fc effector functions are not part ofthe mepolizumab Mechanism Of Action (MOA). Based on the MOA and PKproperties of mepolizumab, there are two functions involved in thebiological activity of this antibody: binding to IL-5 via the CDRs, andbinding to FcRn receptor via the Fc region.

Through the extensive characterization studies performed above and asset out below, it was determined that at least deamidaton, oxidation,and aggregation can lead to antibody variants in the composition ofmepolizumab, and that these variants can impact the function ofmepolizumab. Specific levels of these variants should be maintained toensure appropriate biological function. Function is herein describedwithin the acceptable range of 0.70-1.30 specific antigen bindingactivity (IL5-binding) and 70%-130% FcRn binding. Thus the steps toidentify antibody variants that impact function include: (i) is theantibody variant formed, (ii) does the variant have an impact onfunction, and (iii) what level of variant can result in a functionalcomposition.

Function

IL-5 binding: A statistical analysis was performed to calculate theacceptable antigen binding functional activity range using all drugsubstance (DS) and drug product (DP) release and stability datagenerated to date. The calculated statistical range was compared toclinical experience and evaluated based on the known impact of productrelated variants on potency. Based on this analysis, the acceptableantigen binding functional activity range at time of release and at theend of the shelf-life is specific antigen binding of 0.70-1.30.

The IL-5 specific binding was determined by Surface Plasmon Resonance(SPR) using a BIACORE™ instrument, performed in the binding mode. ThisSPR assay is able to detect decreases in antigen binding that resultfrom changes in mepolizumab and mepolizumab variants linked to potency.

SPR is used to determine the specific antigen binding activity ofmepolizumab. First, mepolizumab reference standard is injected over thesurface of a CM5 sensor chip containing immobilized Protein A and thendiluted IL-5 protein at a fixed concentration is injected, enabling theIL-5 to bind to the captured mepolizumab sample. The concentration ofmepolizumab bound to IL-5, reported as functional binding of mepolizumabto IL-5, is determined from a corresponding mepolizumab referencestandard calibration curve. The SPR result was reported as thefunctional binding concentration of mepolizumab to IL-5, divided by thetotal protein concentration.

FcRn binding: The Neonatal Fc (FcRn) Receptor Binding activity ofmepolizumab was also measured by Surface Plasmon Resonance (SPR) using aBIACORE™ instrument. The acceptable FcRn binding functional activityrange was determined to be 70-130%, based on results generated to dateduring mepolizumab product development, known assay variables, andresults generated for similar mAb products.

The Fc region of mepolizumab binds to FcRn, and this interactionreflects the long serum half-life of mepolizumab (mean terminalhalf-life=20 days). In the SPR assay, a nitrilotriacetic acid (NTA)sensor chip containing immobilized FcRn receptors was used to capture afixed concentration of mepolizumab. First, Ni²⁺ was injected at a fixedconcentration and captured on a NTA sensor chip by chelation of Ni²⁺through NTA. Second, FcRn receptor was injected at a fixed concentrationand the 6× histidine tag at the C-terminus of the alpha chain of theFcRn receptor binds to the Ni²⁺ that had been previously captured.Mepolizumab that had been diluted within the standard curveconcentration range was then injected over the surface of the NTA sensorchip containing captured FcRn receptor. The concentration of mepolizumabbound to the FcRn receptor was extrapolated from a correspondingmepolizumab reference standard calibration curve. The SPR result wasreported as the functional binding concentration of mepolizumab to theFcRn receptor, divided by the total protein concentration.

The SPR method for specific antigen binding and FcRn binding uses areference standard of mepolizumab. The mepolizumab reference standard issimply used in assays to obtain system suitability and samplecomparability data, to ensure methods are performing appropriately. Thereference standard allows the establishment of a calibration curve andconcentrations of the samples are interpolated from the curve.

Acidic Variants

Forced degradation studies were then performed to determine the impactof increased levels of acidic variants, for example deamidation, onantibody function/efficacy, i.e., antigen binding and FcRn bindingactivities.

In pH 9.0 forced degradation studies the composition was adjusted to pH9.0 with 6N sodium hydroxide and was incubated for 30 days at 40° C.Samples were collected at 0, 3, 7, 14 and 30 days and were compared withthe unstressed composition, which was used as a control. The pH 9.0stressed samples were then analyzed by cIEF. The results are shown inTable 10 and FIG. 1 (day 0 and day 3). The pH 9.0 stressed compositionwas degraded beyond the capabilities of the cIEF assay at Day 14;therefore, only results up to the day 7 time point are shown in Table10. At a stressed condition of pH 9.0 for 3 days, the total acidicregion was observed to be 74.4% and 71.9% for two different batches ofthe composition.

TABLE 10 cIEF data summary for pH 9.0 forced degradation studies.Primary Area (%) Manufacturing Total Total Condition Process/Batch DayMain Peak Acidic Basic Control 62.9 35.9 1.2 Elevated pH MDS1 0 62.536.1 1.4 9.0 T004L003S 3 24.8 74.4 0.8 7 8.3 91.7 0.0 MDS2 0 63.7 33.33.0 T0413010 3 26.9 71.9 1.2 7 11.4 88.3 0.3

The pH 9.0 forced degradation study samples of the composition fromdifferent batches were then tested for specific antigen binding activity(Table 11) and FcRn binding activity (Table 12) using standard surfaceplasmon resonance (SPR) methods.

TABLE 11 Data summary of specific antigen binding activity (e.g., humanIL-5 binding activity) measured by SPR in pH 9.0 forced degradationstudy samples of the composition from different batches. PrimaryManufacturing Specific Antigen Condition Process/Batch Day BindingActivity Elevated pH 9.0 MDS1 T04L003S 0 0.96 3 0.74 7 0.60 MDS2T0413010 0 0.94 3 0.74 7 0.62

TABLE 12 FcRn binding measured by SPR in pH 9.0 forced degradation studysamples of the composition from different batches. Primary FcRnManufacturing Binding Condition Process/Batch Day (%) Elevated pH 9.0MDS1 T04L003S 0 91 3 84 7 82 MDS2 T0413010 0 86 3 82 7 80The IL5 specific binding activity at Day 3 (i.e., about 72-74% acidicvariant) was 0.74 for both batches of the composition subjected to pH9.0 forced degradation. The FcRn binding activities were 82% and 80%respectively for both batches of the composition subjected to pH 9.0forced degradation. These values were within the acceptance criteria foreach assay. The acceptance criterion for specific antigen bindingactivity is 0.70-1.30 and the acceptance criterion for FcRn binding is70%-130%. Thus, acidic variant can be as high as about 74% to maintainfunction of the mepolizumab composition.

Deamidation

Forced degradation studies can determine which residues that appear tobe susceptible to deamidation actually deamidate, and whether thedeamidated variant has an impact on function, and what levels ofdeamidation are acceptable within a composition to maintain function.The asparagine residues which were experimentally determined to besusceptible to deamidation are HC N317, HC N386, HC N299, and LC N31.Forced degradation studies were performed to determine the impact ofincreased levels of deamidation LC N31 in the antibody light chain aminoacid sequence shown in SEQ ID NO: 2 on antigen binding activity. Inthese studies the composition from different batches was adjusted to pH9.0 with 6N sodium hydroxide and was incubated for 30 days at 40° C.Samples were collected at 0, 3, 7, 14 and 30 days and were compared withunstressed composition (e.g., reference standard) which was used as acontrol. The pH 9.0 stressed samples were tested by peptide mappingLC-MS/MS. The results are shown in Table 13. When mepolizumab is held atpH 9.0 for 3 days the level of deamidated LC N31 in the antibody lightchain amino acid sequence shown in SEQ ID NO: 2 is 17.4% and 16.8% fordifferent batches of the composition. See Table 13. Antigen and FcRNbinding data for mepolizumab held at pH 9.0 for 3 days are presented inTable 11 and Table 12.

TABLE 13 Percentage deamidation by peptide mapping LC-MS/MS in pH 9.0forced degradation study samples of the composition from differentbatches at day 3. Primary Manufacturing Deamidation (%) ConditionProcess/Batch HC N317 HC N386 LC N31 HC N299 control 0.8 5.5 5.2 0.2Elevated MDS1 0.9 28.2 17.4 1.3 pH 9.0 T04L003S MDS2 1.0 27.8 16.8 1.3T0413010Therefore at Day 3, the specific antigen binding activity of 0.74 (Table11), and FcRn binding activity of 84% and 82% (Table 12), show thatdeamidation at N31 of up to around 17%, and deamidation at N386 of up toaround 28% (Table 13), can maintain a functional composition within theacceptable range of 0.70-1.30 specific antigen binding activity and70%-130% FcRn binding.

Oxidation

Forced degradation studies were performed to experimentally examine thesusceptibility of methionine and other amino acid residues in theantibody heavy and light chains of the composition to oxidation. Forceddegradation studies can determine which residues that appear to besusceptible to oxidation actually oxidize, and whether the oxidizedvariant has an impact on function, and what levels of oxidation areacceptable within a composition to maintain function/efficacy (antigenbinding and/or FcRn binding).

Samples of the composition were incubated with 0.1% hydrogen peroxide(H₂O₂) for 48 hours at room temperature (RT) to induce oxidation.Samples were collected at 0, 6, 12, 24, and 48 hours. These werecompared with unstressed composition (e.g., reference standard) whichwas used as a control. It was determined from these studies themethionine (M) residues most prone to oxidation include HC M64, HC M82,HC M85, HC M254, HC M360, HC M430 of the antibody heavy chain amino acidsequence shown in SEQ ID NO: 1 and LC M4 of the antibody light chainamino acid sequence shown in SEQ ID NO: 2. The methionine (M) residuesmost prone to oxidation include M64, which is located in the HC CDR2;M254 and M430, which are located in the FcRn and Protein A bindingpocket in the Fc region; and M360 of the antibody heavy chain amino acidsequence shown in SEQ ID NO: 1. Methionine residues prone to oxidationto a lesser extent included HC M4, HC M82, and HC M85 of the antibodyheavy chain amino acid sequence shown in SEQ ID NO: 1. In addition, LCC220 of the antibody light chain amino acid sequence shown in SEQ ID NO:2 was determined to be prone to oxidation under chemically inducedconditions. Importantly, LC C220 and HC C222 form the inter-chaindisulfide bond that joins the heavy and light chains.

The levels of sulfoxide resulting from methionine oxidation and ofsulfonic acid resulting from cysteine oxidation were measured usingpeptide mapping LC-MS/MS and is summarized in Table 14. HC M64, M254,M360, and M430 were more than 70% oxidized at 48 hours after incubationwith 0.1% hydrogen peroxide (H₂O₂).

TABLE 14 Percentage oxidation determined by peptide mapping LC-MS/MS instudy samples of the composition from different batches after treatmentwith H₂O₂ for 48 hours. Primary Oxidation (%) Manu- HC facturing HC HCHC HC M82 LC LC Process BDS Batch M64 M254 M360 M430 and 85 M4 C220Control  0.9  3.5  0.5  0.5 0.3 0.2 0.0 MDS1 T04L003S 72.9 99.9 98.595.6 3.2 0.8 7.6 MDS2 T0413010 85.6 99.8 98.1 98.7 3.5 1.0 7.2

Specific antigen binding by the antibodies in the composition wasmeasured by SPR. This demonstrated a greater decrease in specificantigen binding activity at 48 hours in different H₂O₂ stressed batchesas summarized in Table 15. This decrease in antigen binding correlateswith the relatively higher levels of M64 oxidation observed by peptidemapping LC-MS/MS. The specific antigen binding activities in the testedsamples from the different batches of the composition decreased byapproximately 15% and 32%, respectively. When mepolizumab was 85.6%oxidized specific antigen binding activity was still retained at 0.57.The linear relationship between the cumulative levels of oxidation inmepolizumab and specific antigen binding activity was used and it wasdetermined, at worst case, that HC M64 could be approximately 50%oxidized and the antibodies in the composition would still retain theantigen binding activity in the range of 0.70-1.30.

TABLE 15 Specific antigen binding activity measured by SPR in differentbatches of the composition treated with H₂O₂ for 48 hours. PrimaryManufacturing Time Specific Antigen Process/Batch Condition (hours)Binding Activity MDS1 Oxidation 0 0.92 T04L003S Control 48 0.92 0.1%H₂O₂ 0 0.91 48 0.76 MDS2 Oxidation 0 0.96 T0413010 Control 48 0.96 0.1%H₂O₂ 0 0.89 48 0.57The FcRn binding activity profiles of H₂O₂ stressed samples fromdifferent batches of the composition were highly similar with asubstantial decrease in the FcRn binding activity at 48 hours comparedwith the controls (untreated reference standard) as shown in Table 16.HC M254 and HC M430 are located in the Fc region and when oxidized havebeen shown to result in a decrease in FcRn binding. Based on peptidemapping results generated during the forced degradation studies, whenthe composition is chemically oxidized with H₂O₂ for 48 hours, thelevels of oxidized HC M254 and HC M430 observed in the different batchesis ≥95%. The FcRn binding results showed approximately an 80% decreasein antigen FcRn binding in the H₂O₂ stressed samples from differentbatches of the composition. When mepolizumab was ≥90% oxidized FcRnbinding activity was still retained at 22%. The linear relationshipbetween the cumulative levels of oxidation in mepolizumab and FcRnbinding activity was used and it was determined, at worst case, the HCM254 and HC M430 could be 50% oxidized and the antibodies in thecomposition would still retain the FcRn binding activity in the range of70%-130%.

TABLE 16 FcRn binding activity measured by SPR in different batches ofthe composition treated with H₂O₂ for 48 hours. Primary FcRnManufacturing Time Binding Activity Process/Batch Condition (hours) (%)MDS1 Oxidation 0 97 T04L003S Control 48 94 0.1% H₂O₂ 0 79 48 22 MDS2Oxidation 0 97 T0413010 Control 48 98 0.1% H₂O₂ 0 77 48 17

A photo stress study was conducted to determine the impact of lightinduced tryptophan oxidation on the antigen binding activity of theantibodies in different batches of the composition. This showedtryptophan W52 in the antibody heavy chain is prone to oxidation. Forthese studies the composition from different batches was exposed to 1.8million lux-hr of visible light over approximately 60 hours at 25° C. toinduce photo stress. Samples collected at 0, 3, 7, 14, and 30 hours werecompared with an unstressed reference standard of the composition whichwas used as a control. The levels of di-oxidation/kynureninie resultingfrom tryptophan oxidation were highly similar in the different batchesof light exposure stressed composition as summarized in Table 17.Increases in HC W52 oxidation were detected after 60 hours of lightexposure.

TABLE 17 Percentage oxidation measured by peptide mapping LC-MS/MS indifferent batches of the composition after light exposure stress for 60hours. Oxidation Level (%) Primary Manufacturing W52 W52 Process/Batch(+32 Da) (+4 Da) control 0.1 0.0 MDS1 T04L003S 3.3 3.4 MDS2 T0413010 3.54.6

Specific antigen binding activity profiles of the antibodies in thedifferent batches of light exposure stressed composition showed adecrease in specific antigen binding activity over time. This issummarized in Table 18. When mepolizumab was approximately 7% oxidizedspecific antigen binding activity was still retained at 0.53. The linearrelationship between the cumulative levels of tryptophan oxidation inmepolizumab and specific antigen binding activity was used and it wasdetermined, at worst case, W52 could be 3% oxidized and the antibodiesin the composition would still retain the antigen binding activity inthe range of 0.70-1.30.

TABLE 18 Specific Antigen Binding Activity measured by SPR in differentbatches of the composition after light exposure stress for 60 hours.Primary Manufacturing Specific Antigen Process/Batch Condition DayBinding Activity MDS1 T04L003S Light Exposure 0 0.89 Control 60 0.89Light Exposed 0 0.89 60 0.53 MDS2 T0413010 Light Exposure 0 0.93 Control60 0.93 Light Exposed 0 0.93 60 0.55Thus, to maintain function (IL-5 binding, and/or FcRn binding), HC M64could be up to 50% oxidized, HC M254 and HC M430 could be up to 50%oxidized, and W52 could be up to 3% oxidized.

Aggregation

The size distribution of the antibodies in the composition was monitoredby using standard non-denaturing size exclusion chromatography (SEC)methods. Three peaks were detected in the RS composition SEC profile asshown in FIG. 3 and FIG. 4. A main peak at 7.9 minutes with a relativepercentage area of 99.4% was identified as monomer; a minor peak atapproximately 6.7 minutes with a relative percentage area of 0.5% wasidentified as aggregate. A second minor peak has been observed in somebatches, eluting after the main peak which indicates the presence offragment. Typically this peak is below the SEC assay QL of 0.1.

The aggregate peak was further characterized using SEC with multi-anglelight scattering (MALS) detection and analytical ultracentrifugation(AUC). Results show that the SEC-MALS profiles contain an early elutingpeak (dimer) and a later eluting peak (monomer) as shown in FIG. 5 forthe RS composition and FIG. 6 for a different batch of the composition.The line that cross-sects each peak represents the molar mass of thedetected species and the position of the dimer peak, which is notreadily visible in the chromatograms due to the low abundance of dimerin the samples.

The SEC-MALS data was used to calculate the molar mass of the antibodymonomers and dimers. The resulting molar mass of the monomers in the RScomposition and for a different batch of the composition was comparableto the mepolizumab monomer theoretical mass of 148,760 kDa as shown inTable 19. Variability was detected in the molecular weight observed forthe dimer due to the low level of this species present in the sample.

TABLE 19 SEC-MALS analysis of the monomers in the RS composition and fora different batch of the composition. Molar mass (kDa) MepolizumabSample Monomer Dimer Reference Standard 147 304 RS 101245722 BatchT0413010 147 340

Sedimentation velocity area under the curve (AUC) integral analysis wasused as a complementary technique to fraction based methods includingSEC to confirm there is no perturbation of self association equilibriumor exclusion of higher order aggregate from the chromatographicseparation. The results of AUC analysis demonstrated the c(s)distribution contains one dominant species (main peak), identified asmonomer, with a sedimentation coefficient for both the RS batch of thecomposition and another batch of the composition of 2.81 S; and oneaggregate peak, identified as dimer, with a sedimentation coefficient of4.87 S for the RS batch of the composition and 5.10 S for another batchof the composition as shown in Table 20. The difference in sedimentationcoefficient values between the PRS and BDS is not considered substantialand is attributed to the low abundance of dimer within the samples. Theonly high molecular weight species detected was dimer, which isconsistent with SEC-UV and SEC-MALS results.

TABLE 20 AUC Analysis of different batches of the compositionMepolizumab Sedimentation Molecular Weight Sample Coefficient kDaAbundance (n = 3) Monomer Dimer Monomer Dimer Monomer Dimer PRS 2.814.87 137 336 99.1% 0.9% 101245722 BDS 2.81 5.10 136 353 99.3% 0.7%T0413003

The sedimentation coefficients determined for monomer and dimer werelower than the traditionally observed values for IgG₁ monoclonalantibodies. The formulation of mepolizumab contains 12% (w/v) sucrose,resulting in a highly viscous sample which causes the lowersedimentation coefficients observed for these antibody molecules in thecomposition. The results of the SEC-UV, SEC-MALS, and AUC analysis showthat the aggregate species in the composition is antibody dimer.

To investigate the impact of aggregate on antigen binding activity, alow pH study was conducted on the composition from different batches.Composition samples from the different batches were adjusted to pH 3.5with 5N hydrochloric acid. The pH 3.5 adjusted samples were thenincubated for 30 days at 40° C. to induce chemical modifications.Samples collected at 0, 3, 7, 14 and 30 days were compared with anunstressed RS sample of the composition, which was used as a control.This showed when the composition is chemically stressed at low pH 3.5,aggregation is one of the primary degradation pathways.

SEC degradation profiles of pH 3.5 stressed samples of the compositionare shown in FIG. 7 and summarized in Table 21. Under low pH 3.5 stressconditions, different rates of aggregation where observed between thedifferent batches of composition. However, by day 30 both batches of thecomposition reach equilibrium and exhibit similar levels of aggregation.This difference in aggregation rate between the different batches isattributed to the higher level of covalent dimer versus non-covalentdimer in the batches. The slower aggregation rate observed in the MDS1batch of the composition is attributed to the higher proportion ofnon-covalent dimer relative to that of the MDS2 batch; non-covalentdimer associates and dissociates until equilibrium is reached, which mayslow the overall rate of aggregate formation.

TABLE 21 SEC data summary for an untreated RS batch of the compositionand pH stressed batches of the composition. Primary Manufacturing Area(%) Condition Process/Batch Day Monomer Aggregate Fragment control 99.60.4 0.0 Low pH 3.5 MDS1 0 99.0 1.0 0.0 T004L003S 3 81.4 14.3 4.2 7 59.335.4 5.2 14 47.9 44.4 7.7 30 36.8 51.2 12.0 MDS2 0 98.4 1.6 0.1 T04130103 56.5 40.5 3.1 7 48.3 46.9 4.8 14 41.9 50.7 7.4 30 34.0 54.4 11.5

Specific antigen binding activity profiles of samples of the pH 3.5stressed batches of the composition showed a decrease in specificantigen binding activity over time as summarized in Table 22.

TABLE 22 Data summary of specific antigen binding activity measured bySPR in samples of pH stressed batches of the composition. PrimarySpecific Antigen Manufacturing Binding Activity Condition Process/BatchDay (mg/mL) Low pH 3.5 MDS1 T04L003S 0 0.92 3 0.56 7 0.43 MDS2 T04130100 0.95 3 0.57 7 0.43

The FcRn binding activity profiles of samples of the pH 3.5 stressedbatches of the composition showed a decrease in FcRn binding activityover time as summarized in Table 23.

TABLE 23 Data summary of FcRn binding measured by SPR in pH stressedbatches of the composition. Primary FcRn Manufacturing Binding ConditionProcess/Batch Day (%) Low pH 3.5 MDS1 T04L003S 0 90 3 54 7 46 MDS2T0413010 0 86 3 51 7 43

In summary, Tables 21-23 show that there is approximately a 50% decreasein antigen binding and FcRn binding when there is approximately 40%aggregate present in the sample. When mepolizumab was approximately 40%aggregated, specific antigen binding activity was still retained at 0.57and FcRn binding activity was retained at 51% (Table 22 MDS2 Day 3).There was a slightly different degradation profile for aggregate contentbetween MDS1 and MDS2 at Day 3 (Table 21) because of the differentratios of covalent versus non-covalent dimer.

The linear relationship between the aggregation in mepolizumab andspecific antigen and FcRn binding activities was used from MDS2 and itwas determined, at worst case, mepolizumab could be 20% aggregate andthe antibodies in the composition would still retain the antigen bindingactivity in the range of 0.70-1.30 and FcRn binding activity of 70-130%.

Therefore, it is possible for the antibodies in the compositioncomprising mepolizumab to be 20% aggregated and still retain IL-5binding activity in the range of 0.70-1.30 and FcRn binding activity inthe range of 70%-130%.

HCP

Residual CHO host cell protein levels in the mepolizumab composition aremeasured using an enzyme-linked immunosorbent assay (ELISA). This methoduses antibodies produced against native antigens of the CHO cell linegrown under conditions that mimic the production process conditions ofmepolizumab.

It was determined that for the mepolizumab composition, an acceptablerange for HCP content is ≤10 ng/mg. This range is derived from releasedata generated to date and represents the true analytical and processvariability. 37 different batches of drug substance had the followingHCP content: 1.1 ng/mg (2 batches), 1.0 ng/mg (5 batches), 0.9 ng/mg (1batch), 0.8 ng/mg (3 batches), 0.7 ng/mg (1 batch), 0.6 (1 batch), 0.5ng/mg (1 batch), ≤0.5 ng/mg (4 batches), ≤1 ng/mg (19 batches).

In summary, there are two predominant functions involved in thebiological activity of mepolizumab: binding to IL-5 via the CDRs, andbinding to FcRn receptor via the Fc region. Through the extensivecharacterization studies performed above, it was determined thatparticular deamidated antibody variants, particular oxidated antibodyvariants, and aggregated antibody variants, can impact the function ofthe composition of mepolizumab. Therefore, specific levels of thesevariants should be maintained to ensure appropriate function/efficacy.

Example 3 Informal Sequence Listing

Underlining below identifies CDR sequences, according to the Kabatdefinition of CDRs, in the variable heavy and variable light chainportions of the antibodies or the nucleic acid sequences encoding theseCDR sequences. For example, in SEQ ID NO: 1 the frameworks and CDRs arepresented as plaintext framework1, underlined CDR1, plaintextframework2, underlined CDR2, plaintext framework3, underlined CDR3 andplaintext framework4 in order from the amino proximal portion to thecarboxy terminal portion of the sequences presented. Asterisks to theright of a character for a single letter amino acid code indicates theamino acid residue to the left is a N-glycosylation site. This scheme isused in SEQ ID NOs: 1-4, 11, 12 and 19-22, etc. for example Aminoterminal methionine residues shown in these sequences can be cleaved.Thus, the sequences here showing an amino terminal methionine residueshould also be considered to disclose the cleaved versions of theseproteins lacking such an amino terminal methionine residue. Nucleicacids sequences are presented as DNA nucleic acid sequences and include“t” nucleic acid residues, the corresponding RNA sequence should also beconsidered as disclosed such that “t” nucleic acid residues may also beregarded as disclosing a “u” nucleic acid residue. Additionally, the 5′proximal “atg” start codon and the 3′ proximal “taa,” “tag,” and “tga”stop codons have been omitted from the cDNA nucleic acid sequencesbelow. This is the case for SEQ ID NOs: 31-34, etc. for example.

MEPOLIZUMAB FULL LENGTH HEAVY CHAIN SEQ ID NO: 1QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSALMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKMEPOLIZUMAB FULL LENGTH LIGHT CHAIN SEQ ID NO: 2DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MEPOLIZUMAB VHSEQ ID NO: 3 QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSALMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGT PVTVSSMEPOLIZUMAB VL SEQ ID NO: 4DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEIKMEPOLIZUMAB CDRH1 SEQ ID NO: 5 SYSVH MEPOLIZUMAB CDRH2 SEQ ID NO: 6VIWASGGTDYNSALMS MEPOLIZUMAB CDRH3 SEQ ID NO: 7 DPPSSLLRLDYMEPOLIZUMAB CDRL1 SEQ ID NO: 8 KSSQSLLNSGNQKNYLA MEPOLIZUMAB CDRL2SEQ ID NO: 9 GASTRES MEPOLIZUMAB CDRL3 SEQ ID NO: 10 QNVHSFPFTHUMAN IL-5 (MATURE PROTEIN) SEQ ID NO: 11IPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQEFLGVMNTEWIIESHUMAN IL-5 RECEPTOR SUBUNIT ALPHA ISOFORM 1 (MATURE PROTEIN)SEQ ID NO: 12 DLLPDEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTSIVNLTCTTNTTEDNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGRNIACWFPRTFILSKGRDWLAVLVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSIQWEKPVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLILSLICKICHLWIKLFPPIPAPKSNIKDLFVTTNYEKAGSSETEIEVICYIEKPGVETLEDSVFDNA ENCODING MEPOLIZUMAB FULL LENGTH HEAVY CHAIN SEQ ID NO: 13caggttaccctgcgtgaatccggtccggcactagttaaaccgacccagaccctgacgttaacctgcaccgtctccggtttctccctgacgagctatagtgtacactgggtccgtcagccgccgggtaaaggtctagaatggctgggtgtaatatgggctagtggaggcacagattataattcggctctcatgtcccgtctgtcgatatccaaagacacctcccgtaaccaggttgttctgaccatgactaacatggacccggttgacaccgctacctactactgcgctcgagatcccccttcttccttactacggcttgactactggggtcgtggtaccccagttaccgtgagctcagctagtaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaagDNA ENCODING MEPOLIZUMAB FULL LENGTH LIGHT CHAIN SEQ ID NO: 14gatatcgtgatgacccagtctccagactcgctagctgtgtctctgggcgagagggccaccatcaactgcaagagctctcagagtctgttaaacagtggaaatcaaaagaactacttggcctggtatcagcagaaacccgggcagcctcctaagagctcatttacggggcgtcgactagggaatctggggtacctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtatactactgtcagaatgttcatagttttccattcacgttcggcggagggaccaagttggagatcaaacgtactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

The present invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

1.-19. (canceled)
 20. A composition comprising: (i) an anti-IL-5antibody comprising a heavy chain amino acid sequence as shown in SEQ IDNO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2;and, (ii) an oxidized anti-IL-5 antibody variant comprising SEQ ID NO:1and SEQ ID NO: 2, except that residue M64 of SEQ ID NO:1 is oxidized.21. The composition according to claim 20, wherein the M64 oxidizedantibody variant of (ii) comprises from 0.5% to 50% of the anti-IL5antibodies in the composition.
 22. The composition of claim 20, whereinthe composition comprises an oxidized variant of SEQ ID NO: 1, whereresidue W52 is oxidized.
 23. The composition according to claim 22,wherein the W52 oxidized antibody variant of SEQ ID NO:1 comprises from0% to 3% of the anti-IL-5 antibodies in the composition.
 24. Thecomposition according to claim 20, wherein the composition comprises adeamidated variant of SEQ ID NO: 2, where residue N31 is deamidated toaspartic acid or iso-aspartic acid.
 25. The composition according toclaim 24, wherein the N31 deamidated antibody variant of SEQ ID NO:2comprises from 0% to 20% of the anti-IL-5 antibodies in the composition.26. The composition according to claim 25, wherein the N31 deamidatedantibody variant of SEQ ID NO:2 comprises ≤17.5%, ≤15%, ≤12.5, ≤10%, or≤7.5% of the anti-IL-5 antibodies in the composition.
 27. Thecomposition according to claim 20, wherein the composition comprises adeamidated variant of SEQ ID NO: 1, where residue N386 is deamidated toaspartic acid or iso-aspartic acid.
 28. The composition according toclaim 27, wherein the N386 deamidated antibody variant of SEQ ID NO:1comprises from 0% to 35% of the anti-IL-5 antibodies in the composition.29. The composition according to claim 20, wherein the compositioncomprises an oxidized antibody variant of SEQ ID NO:1, where residueM254 is oxidized, M360 is oxidized, and M430 is oxidized.
 30. Thecomposition according to claim 29, wherein the M254, M360, and M430oxidized variants comprise from 0% to 50% of the anti-IL-5 antibodies inthe composition
 31. The composition according to claim 20, wherein thecomposition comprises ≥50% SEQ ID NO: 1 C-terminal lysine K449 deletedantibody variant.
 32. The composition according to claim 20, wherein thecomposition comprises: ≥50% SEQ ID NO: 1 pyro-glutamate N-terminalantibody variant.
 33. The composition according to claim 20, wherein thecomposition comprises ≤80% acidic antibody variants.
 34. The compositionaccording to claim 20, wherein the composition comprises ≤20% aggregatedantibody variant.
 35. The composition according to claim 20, wherein thecomposition has at least 0.70 IL-5 specific antigen binding activitycompared with a reference standard composition, the reference standardcomposition comprising: (a) an antibody having a heavy chain amino acidsequence as shown in SEQ ID NO: 1 and a light chain amino acid sequenceas shown in SEQ ID NO: 2; (b) 98% or more SEQ ID NO:1 C-terminal lysinedeleted variant of the antibody; (c) 95% or more SEQ ID NO:1 N-terminalpyroglutamate variant of the antibody; (d) 6% or less of the antibodywherein residue 31 of SEQ ID NO: 2 is replaced with either an asparticacid or an iso-aspartic acid; (e) 4% or less methionine or cysteineoxidated variant; (f) 0.1% or less tryptophan oxidated variant; and (g)0.4% aggregated antibodies of the reference standard.
 36. Apharmaceutical composition comprising a composition according to claim20 and a pharmaceutically acceptable carrier.
 37. The compositionaccording to claim 20, wherein the antibody is at a concentration ofbetween 75 mg/mL to 100 mg/mL.
 38. The composition according to claim20, wherein the composition comprises one or a combination of: i. abuffering agent selected from the group consisting of sodium phosphatedibasic heptahydrate, phosphate, citrate, sodium phosphate, potassiumphosphate, sodium citrate, and histidine, providing a pH of between 6.8and 7.2 or a pH of between 6.2 and 6.6; ii. a sugar; iii. polysorbate80; or iv. EDTA.
 39. The composition according to claim 38, wherein thebuffering agent is present in the range from 10-30 mM.
 40. Thecomposition according to claim 38, wherein the sugar is sucrose and ispresent at a concentration of 5-20% weight by volume.
 41. Thecomposition according to claim 38, wherein the polysorbate 80 is presentat a concentration of 0.01% to 0.1% weight by volume.
 42. Thecomposition according to claim 38, wherein the EDTA is present at aconcentration of 0.01 mM to 0.1 mM.
 43. The composition according toclaim 38, wherein the composition comprises 100 mg/ml antibody and; i)15.5 mM sodium phosphate dibasic heptahydrate and 4.5 mM citric acidmonohydrate at pH 6.3; ii) 12% weight of sucrose to volume; iii) 0.02%weight of polysorbate 80 to volume, and iv) 0.05 mM EDTA.
 44. Thecomposition according to claim 38, wherein the composition islyophilized and comprises 100 mg/mL antibody and; i) 20 mM sodiumphosphate dibasic heptahydrate at pH about 6.8 to about 7.2; ii) 12%weight of sucrose to volume; and iii) 0.05% weight of polysorbate 80 tovolume.